Analyte determination utilizing mass tagging reagents comprising a non-encoded detectable label

ABSTRACT

This invention pertains to methods, mixtures, kits and compositions pertaining to analyte determination and/or quantification by mass spectrometry using compounds comprising a reporter moiety and a non-encoded detectable label. The compounds can be used in sets for the analysis of mixtures of labeled analytes.

PRIORITY AND RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/818,007 filed Jun. 30, 2006, incorporated hereinby reference.

The section headings used herein are for organizational purposes onlyand should not be construed as limiting the subject matter describedherein in any way.

FIELD

This invention pertains to methods, mixtures, kits and compositionspertaining to analyte determination using mass spectrometry techniques.

INTRODUCTION

This invention pertains the determination of an analyte or analytes bymass analysis. In various embodiments, the mass spectrometric analysiscan be coupled with an electrophoretic separations technique. An analytecan be any molecule of interest. Non-limiting examples of analytes(natural and/or synthetic) include, but are not limited to, proteins,peptides, nucleic acids, carbohydrates, lipids, amino acids, vitamins,steroids, prostaglandins and/or other small molecules having a mass ofless than 1500 daltons (Da). Analytes can be determined using uniquelabeling reagents that permit the relative and/or absolutequantification of the analytes in complex mixtures. The labelingreagents can be used in isomeric and/or isobaric sets for the analysisof complex sample mixtures wherein the labeling reagents can be isomericand/or isobaric.

Isomeric and/or isobaric labeling reagents comprising a reporter moiety,a balance (or linker) moiety and a reactive group, wherein the reactivegroup can be substituted by the analyte in the analyte reacted form ofthe composition, are known. Examples of labeling reagents and labeledanalytes comprising these three elements have been disclosed in, forexample, published copending and commonly owned U.S. Patent ApplicationSerial Nos. US 2004-0219685 A1, US 2005-0114042 A1, US 2005-0147982 A1,US 2005-0147985 A1, US 2005-0147987 A1, US 2005-0148771 A1, US2005-0148773 A1 and US 2005-0148774 A1. Sets of isomeric and/or isobariclabeling reagents can be used to label, for example, the analytes of twoor more different samples wherein the different labeling reagents of aset all have the same gross mass but wherein each reporter moiety can beuniquely encoded such that each reporter moiety of the set has a uniquegross mass and produces a corresponding signature ion of unique grossmass. Because all the reagents of the set can have the same gross massbut can comprise a reporter moiety of unique gross mass, the balance (orlinker) will generally (but not necessarily) also comprise one or moreheavy atom isotopes to thereby “balance” the mass of each uniquereporter such that the reporter/linker combination of each labelingreagent of the set has the same gross mass.

Described herein are new labeling reagents that can be used in isomericand/or isobaric sets, wherein the labeling reagents comprise anon-encoded detectable label. FIG. 1 a illustrates the elements ofexemplary labeling reagents (and labeled analytes) of this invention andsome information about possible fragmentation characteristics of themolecules in a mass spectrometer. As illustrated, the linker moiety caneither connect the elements of the labeling reagent (or labeled analyte)in a linear or a branched mode. When used in isomeric and/or isobaricsets, various non-encoded detectable labels of the labeling reagents ofa set can all: 1) be independently detectable; 2) comprise the samegross mass; and 3) comprise the same net charge. Accordingly, thelabeling reagents disclosed herein can comprise both a moiety that canbe independently detected by non-mass spectrometry methods as well as adifferent moiety that can be independently detected in a massspectrometer. In particular, the novel labeling reagents describedherein can be used, for example, in electrophoretic separationsprocesses combined with mass spectrometric analysis wherein theproperties of the non-encoded detectable labels can be used to locateand/or quantify mixtures of labeled analytes during and/or after theelectrophoretic separation and wherein mass spectrometry can be used toidentify and/or quantify the analytes of interest in a sample orsamples, or fractions thereof.

In some embodiments, the labeling reagents and/or labeled analytes canbe support-bound. FIG. 1 b illustrates the elements of exemplarysupport-bound labeling reagents (and support-bound labeled analytes) ofthis invention and some information about the fragmentationcharacteristics of the molecules in a mass spectrometer. As illustrated,the linker moiety can either connect the elements of the support-boundlabeling reagent (or support-bound labeled analyte) in a linear or abranched mode.

Embodiments of this invention are particularly well suited for themultiplex analysis of complex sample mixtures. For example, someembodiments of this invention can be used in proteomic analysis and/orgenomic analysis as well as for correlation studies related to genomicand/or proteomic analysis. Some embodiments of this invention can beused for small molecule analysis, such as for lipid, steroid, vitamin,prostaglandins and/or amino acid analysis. Experimental analysis forwhich the isomeric and/or isobaric reagents can be used includes, but isnot limited to, time course studies, biomarker analysis, multiplexproteomic analysis, mudpit experiments, affinity pull-downs,determination of post-translational modifications (PTMs) (see forexample published United States Patent Application No. US 2005-0208550A1) and multiple control experiments.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 a is an illustration of the various fragmentation characteristicsof exemplary labeling reagents represented in the form of theirelements.

FIG. 1 b is an illustration of the certain fragmentation characteristicsof exemplary support-bound labeling reagents represented in the form oftheir elements.

FIG. 2 a is an illustration of the certain fragmentation characteristicsof various labeling reagents represented by a generic formula.

FIG. 2 b is an illustration of the certain fragmentation characteristicsof various labeling reagents.

FIG. 2 c is an illustration of the certain fragmentation characteristicsof various labeling reagents.

FIG. 3 a is an illustration of the certain fragmentation characteristicsof various labeled analytes represented by a generic formula.

FIG. 3 b is an illustration of the certain fragmentation characteristicsof various labeled analytes.

FIG. 3 c is an illustration of the certain fragmentation characteristicsof various support-bound labeled analytes.

FIG. 3 d is an illustration of the certain fragmentation characteristicsof various labeled analytes that have been released from a solidsupport.

FIG. 4 a is an illustration of exemplary isobaric compounds.

FIG. 4 b is an illustration of exemplary isomeric isobaric compounds.

FIG. 5 is an illustration of various possible reporter ions (signatureions) that can be generated from certain N-methyl piperazine basedreporter moieties.

FIG. 6 is an illustration of the process of using a support-boundlabeling reagent to prepare a support-bound labeled analyte followed byrelease of the labeled analyte from the solid support.

FIG. 7 is an illustration of a scheme for the synthesis of variousactive esters.

FIG. 8 a is an illustration of a possible isotopic coding strategy forlabeling reagents of formulas II and III.

FIG. 8 b is an illustration of a possible isotopic coding strategy forlabeling reagents of formulas II^(ss) and III^(ss).

FIG. 9 a illustrates part of a prophetic synthetic route to arepresentative labeling reagent.

FIG. 9 b illustrates the remaining part (with respect to FIG. 9 a) of aprophetic synthetic route to a representative labeling reagent.

FIGS. 10 a to 10 d illustrate various steps for the synthesis of anencoded N-Fmoc,N′-methyl-ethylene diamine.

FIG. 11 illustrates the synthesis of a part of an encodedreporter/linker moiety.

FIG. 12 illustrates the synthesis of a part of an encodedreporter/linker moiety.

FIG. 13 a illustrates part of a prophetic synthetic route to arepresentative labeling reagent.

FIG. 13 b illustrates the remaining part (with respect to FIG. 13 a) ofa prophetic synthetic route to a representative labeling reagent.

FIG. 14 illustrates a proposed synthetic route to Non-Encoded DetectableLabels

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference herein in their entirety for any and all purposes.

DEFINITIONS

For the purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example ininterpreting the document where the term is originally used). The use of“or” herein means “and/or” unless stated otherwise or where the use of“and/or” is clearly inappropriate. The use of “a” herein means “one ormore” unless stated otherwise or where the use of “one or more” isclearly inappropriate. The use of “comprise,” “comprises,” “comprising”“include,” “includes,” and “including” are interchangeable and notintended to be limiting. Furthermore, where the description of one ormore embodiments uses the term “comprising,” those skilled in the artwould understand that in some specific instances, the embodiment orembodiments can be alternatively described using language “consistingessentially of” and/or “consisting of.” It should also be understoodthat in some embodiments the order of steps or order for performingcertain actions is immaterial so long as the present teachings remainoperable. Moreover, in some embodiments two or more steps or actions canbe conducted simultaneously.

a.) As used herein, “analyte” refers to any molecule of interest thatmay be determined. Non-limiting examples of analytes include, but arenot limited to, proteins, peptides, nucleic acids (e.g. DNA or RNA),amino acids, carbohydrates, lipids, steroids, vitamins (e.g. vitamin D₂(sometimes referred to as ergocalciferol), vitamin D₃ (sometimesreferred to a cholecalciferol), vitamin B₂, vitamin B₆, vitamin A,vitamin A₂, vitamin E, vitamin K, and metabolites thereof),prostaglandins, and other small molecules with a molecular weight ofless than 1500 daltons (Da). The source of the analyte, or the samplecomprising the analyte, is not a limitation as it can come from anysource. The analyte or analytes can be natural or synthetic.Non-limiting examples of sources for the analyte, or the samplecomprising the analyte, include cells or tissues, or cultures (orsubcultures) thereof. Non-limiting examples of analyte sources include,but are not limited to, crude or processed cell lysates, body fluids,tissue extracts, cell extracts or fractions (or portions) from aseparations process such as a chromatographic separation, a 1Delectrophoretic separation, a 2D electrophoretic separation or acapillary electrophoretic separation. Body fluids include, but are notlimited to, blood, urine, feces, spinal fluid, cerebral fluid, amnioticfluid, lymph fluid or a fluid from a glandular secretion. By processedcell lysate we mean that the cell lysate is treated, in addition to thetreatments needed to lyse the cell, to thereby perform additionalprocessing of the collected material. For example, the sample can be acell lysate comprising one or more analytes that are peptides formed bytreatment of the cell lysate with one or more proteolytic enzymes tothereby digest precursor peptides and/or proteins.

b.) Except as when clearly not intended based upon the context in whichit is being used (e.g. when made in reference to a specific structurethat dictates otherwise), “ester” refers to both an ester and/or athioester.

c.) As used herein, “cleavable linker” refers to a moiety covalentlyattached to a solid support that cleavably links a labeling reagent orlabeled analyte to a support and wherein at least one bond in thecleavable linker can be cleaved by treatment with light, heat orchemical reagent(s) (for the avoidance of doubt, “chemical reagent(s)”is intended to include an enzyme or enzymes) to thereby release thelabel or labeled analyte, or a fragment thereof, from the support.

d.) As used herein, “fragmentation” refers to the breaking of a covalentbond.

e.) As used herein, “fragment” refers to a product of fragmentation(noun) or the operation of causing fragmentation (verb).

f.) As used herein, “hydrate form” refers to any hydration state of acompound or a mixture of more than one hydration states of a compound.For example, a labeling reagent discussed herein can be a hemihydrate, amonohydrate, a dihydrate, etc. Moreover, a sample of a labeling reagentdescribed herein can, for example, simultaneously comprise monohydrate,dihydrate and hemihydrate forms.

g.) As used herein, a halogen group or halide refers to —F (fluorine),—Cl (chlorine), —Br (bromine), or —I (iodine).

h.) As used herein with respect to a compound, “isotopically enriched”refers to a compound (e.g. labeling reagent) that has been enriched withone or more heavy atom isotopes (e.g. stable isotopes such as deuterium(i.e. ²H), ¹³C, ¹⁵N, ¹⁸O, ³⁴S, ³⁷Cl or ⁸¹Br). The isotopically enrichedcompound can be synthetically enriched. In some embodiments, unstableisotopes can also be used (e.g. ¹⁴C or ³H). By “enriched” we mean thatthe amount of heavy atom isotope exceeds natural isotopic abundance. Invarious embodiments, the isotopically enriched compound can comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more isotopically enriched sites.

Because isotopic enrichment is not 100% effective, there can beimpurities in a sample of the compound that are of lesser states ofenrichment and these will have a lower mass. Likewise, because ofover-enrichment (e.g. undesired enrichment) and because of naturalisotopic abundance, there can be impurities in a sample of the compoundthat are of greater mass. In some embodiments, each incorporated heavyatom isotope can be present at an isotopically enriched site in at least80 percent isotopic purity. In some embodiments, each incorporated heavyatom isotope can be present at an isotopically enriched sited in atleast 93 percent isotopic purity. In some embodiments, each incorporatedheavy atom isotope can be present at an isotopically enriched site in atleast 96 percent isotopic purity. In some embodiments, each incorporatedheavy atom isotope can be present at an isotopically enriched site in atleast 98 percent purity.

i.) As used herein, “isotopically enriched site” refers to the positionin a compound where a heavy atom isotope is substituted for a lightversion of the atom (e.g. substitution of ¹³C for ¹²C, ¹⁸O for ¹⁶O, ¹⁵Nfor ¹⁴N or deuterium for hydrogen).

j.) As used herein with respect to a compound, “light” refers to thecompound as not being enriched with a heavy atom isotope. As used hereinwith respect to an atom, “light” refers to the lowest mass isotope ofthe atom. As used herein with respect to a compound, “heavy” refers tothe compound as being enriched with at least one heavy atom isotope. Asused herein with respect to an atom, “heavy” refers to a heavy massisotope of the atom.

k). As used herein, “labeling reagent” refers to a moiety suitable tomark an analyte for determination. The term label is synonymous with theterms tag and mark and other equivalent terms and phrases. For example,a labeled analyte can also be referred to as a tagged analyte or amarked analyte. Accordingly the terms “label”, “tag”, “mass tag”, “mark”and derivatives of these terms, are equivalent and interchangeable andrefer to a moiety suitable to mark, or that has marked, an analyte fordetermination. Sometimes a labeling reagent can be referred to a taggingreagent or a mass tagging reagent.

l.) As used herein, “natural isotopic abundance” refers to the level (ordistribution) of one or more heavy isotopes found in a compound basedupon the natural prevalence of an isotope or isotopes in nature. Forexample, a natural compound obtained from living plant matter maycontain about 1.08% ¹³C relative to ¹²C.

m.) As used herein, isobars are structurally indistinguishable compounds(except for isotopic content and/or distribution of heavy atom isotopes)of the same nominal gross mass. For the avoidance of any doubt,compounds 1-4 of FIG. 4 a are isobaric by the definition set forthherein. By comparison, as used herein isomers are structurallydistinguishable compounds of the same nominal gross mass. Exemplaryisomeric isobars are illustrated in FIG. 4 b.

n.) As used herein, “support”, “solid support”, “solid carrier” or“resin” refers to any solid phase material. Solid support encompassesterms such as “support”, “synthesis support”, “solid phase”, “surface”“membrane” and/or “support”. A solid support can be composed of organicpolymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A solid support can also be inorganic,such as glass, silica, controlled-pore-glass (CPG), or reverse-phasesilica. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression or other container, vessel, feature orlocation. A plurality of solid supports can be configured in an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments.

o.) As used herein, “support-bound” refers to a compound that iscovalently immobilized to a solid support.

p.) As used herein, “sample, or a fraction thereof” or “sample fraction”can be used to refer to a fraction of a sample. The fraction of thesample can be generated either by simply withdrawing a fraction of asample else it can be generated by performing a separations process thatresults in the sample being fractionated into two or more fractions.Unless the content of the description indicates otherwise, these phrasesare equivalent and interchangeable and refer to either type of creationof a fraction (or portion) of a sample.

q.) As used herein, “signature ion” and “reporter ion” areinterchangeable and both refer to the signature ion of unique massproduced from the reporter moiety by fragmentation of a labeling reagentor labeled analyte in a mass spectrometer. The signature ion or reporterion can be used to identify the unique labeling reagent used to label ananalyte and its peak intensity in MS/MS analysis can be correlated withthe amount of labeled analyte present in the sample (or fractionthereof) that is analyzed. As used herein, the signature ion or reporterion is sometimes merely referred to as a reporter. As used herein, thereporter moiety is also sometimes merely referred to a reporter. It isto be understood that the reporter moiety refers to the group attachedto a labeling reagent, labeled analyte or fragment thereof and thereporter ion refers to the fragment ion generated upon fragmentation ofthe bond that links the reporter moiety to the labeling reagent, labeledanalyte or a fragment thereof. Accordingly, the context in which theword “reporter” is used will indicate its intended meaning. It also isto be understood that the phrase “unique reporter moiety” is equivalentto, and interchangeable with, “reporter moiety of unique mass” and that“unique reporter ion” or “unique signature ion” is equivalent to, andinterchangeable with, “reporter ion of unique mass” or “signature ion ofunique mass”.

r.) As used herein, the term “salt form” refers to a salt of a compoundor a mixture of salts of a compound. In addition, zwitterionic forms ofa compound are also included in the term “salt form.” Salts of compoundshaving an amine, or other basic, group can be obtained, for example, byreaction with a suitable organic or inorganic acid, such as hydrogenchloride, hydrogen bromide, acetic acid, perchloric acid and the like(including any combinations thereof). Compounds with a quaternaryammonium group may also contain a counteranion such as chloride,bromide, iodide, acetate, perchlorate and the like (including anycombinations thereof). Salts of compounds having a carboxylic acid, orother acidic functional group, can be prepared by reacting the compoundwith a suitable base, for example, a hydroxide base. Accordingly, saltsof acidic functional groups may have a countercation, such as sodium,potassium, magnesium, calcium, etc. (including any combinationsthereof).

s.) As used herein, the term “steroid” is intended to include, but notbe limited to, cortisol, 11-desoxycortisol (compound S), corticosterone,testosterone, epitestosterone, desoxymethyltestosterone,tetrahydrogestrinone (THG), estradiol, estrone, 4-hydroxyestrone,2-methoxyestrone, 2-hydroxyestrone, 16-ketoestradiol, 16alpha-hydroxyestrone, 2-hydroxyestrone-3-methylether, prednisone,prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA),17 OH pregnenolone, 17 OH progesterone, 17 OH progesterone,androsterone, epiandrosterone, delta 4 androstenedione (D4A),stigmasterol, and cholesterol.

t.) As used herein, “synthetic compound” refers to a compound that iscreated by manipulation of processes including the manipulation ofnaturally occurring pathways. Thus, a synthetic compound can be producedusing synthetic chemistry techniques. However, as used herein,“synthetic compound” is also intended to include compounds that areproduced, for example, by enzymatic methods, including for example,feeding isotopically enriched compounds to organisms, such as bacteriaor yeast, that alter them to thereby produce the isotopically enrichedlabeling reagents, or intermediates of the labeling reagents, describedherein.

u.) As used herein, “synthetically enriched” or “enriched synthetically”refers to the manipulation of a synthetic or natural process to therebyproduce a synthetic compound such as the isotopically enriched labelingreagents, or intermediates to the labeling reagents, described herein.

v.) It is well accepted that the mass of an atom or molecule can beapproximated, often to the nearest whole number atomic mass unit or thenearest tenth or hundredth of an atomic mass unit. As used herein,“gross mass” refers to the absolute mass as well as to the approximatemass within a range where the use of isotopes of different atom typesare so close in mass that they are the functional equivalent for thepurpose of balancing the mass of the reporter and/or linker moieties (sothat the gross mass of the reporter/linker combination is the samewithin a set or kit of isomeric and/or isobaric labeling reagents)whether or not the very small difference in mass of the differentisotopes types used can be detected.

For example, the common isotopes of sulfur have a gross mass of 32(actual mass 31.9720) and 34 (actual mass 33.9678), the common isotopesof oxygen have a gross mass of 16.0 (actual mass 15.9949) and 18.0(actual mass 17.9992), the common isotopes of carbon have a gross massof 12.0 (actual mass 12.00000) and 13.0 (actual mass 13.00336) and thecommon isotopes of nitrogen have a gross mass of 14.0 (actual mass14.0031) and 15.0 (actual mass 15.0001). Whilst these values areapproximate, one of skill in the art will appreciate that if one usesthe ¹⁸O isotope (or the ³⁴S isotope) at an isotopically enriched sitewithin one label of a set, the additional 2 mass units (over the isotopeof oxygen having a gross mass of 16.0) can, for example, be compensatedfor in a different label of the set comprising ¹⁸O (or the ³²S isotope)by incorporating, elsewhere in the label, two carbon ¹³C atoms, insteadof two ¹²C atoms, two ¹⁵N atoms, instead of two ¹⁴N atoms or even one¹³C atom and one ¹⁵N atom, instead of a ¹²C and a ¹⁴N, to compensate forthe ¹⁸O (or the ³⁴S isotope). In this way the two different labels ofthe set can have the same gross mass since the very small actualdifferences in mass between the use of two ¹³C atoms (instead of two ¹²Catoms), two ¹⁵N atoms (instead of two ¹⁴N atoms), one ¹³C and one ¹⁵N(instead of a ¹²C and ¹⁴N) or one ¹⁸O atom (instead of one ¹⁸O atom—orone ³⁴S instead of one ³²S), to thereby achieve an increase in mass oftwo Daltons in all of the labels of the set or kit, is not an impedimentto the nature of the analysis.

This can be illustrated with reference to FIG. 4 a. In FIG. 4 a, thereporter/linker combination of compound 3 (FIG. 4 a; molecular formula:C₅ ¹³CH₁₁ ¹⁵N₂O) has two ¹⁵N atoms and one ¹³C atom and a totaltheoretical mass of 130.085. By comparison, the reporter/linker moietyof isobar 1 (FIG. 4 a; molecular formula C₅ ¹³CH₁₁N₂ ¹⁸O) has one ¹⁸Oatom and one ¹³C atom and a total theoretical mass of 130.095. Compounds1 and 3 can be isobars that are structurally indistinguishable, exceptfor heavy atom isotope content, although there can be a slight absolutemass difference of the reporter/linker moiety (mass 130.095 vs. mass130.085 respectively). However, the gross mass of the reporter/linkermoiety of compounds 1 and 3 is 130.1 for the purposes of this inventionsince this is not an impediment to the analysis whether or not the massspectrometer is sensitive enough to measure the small difference betweenthe absolute mass of the reporter ions generated from isobars 1 and 3.

Similarly with reference to FIG. 4 b, two isomeric reagents areillustrated wherein the mass of the reporter/linker moiety of compounds5 and 6 is 144.111 and 144.100, respectively. The gross mass of thereporter/linker moiety of these compounds is 144.1 for the purposes ofthis invention since it is not an impediment to the analysis whether ornot the mass spectrometer is sensitive enough to measure the smalldifference between the absolute mass of the reporter ions generated fromcompounds 5 and 6.

From FIGS. 4 a and 4 b, it is clear that the distribution of the sameheavy atom isotopes within a structure is not the only consideration forthe creation of sets of isomeric and/or isobaric labeling reagents. Itis possible to mix heavy atom isotope types to achieve isomers and/orisobars of a desired gross mass. In this way, both the selection(combination) of heavy atom isotopes as well as their distribution isavailable for consideration in the production of the isomeric and/orisobaric labeling reagents useful for embodiments of this invention.

w.) As used herein, the term “alkyl” refers to a straight chained orbranched C₂-C₈ hydrocarbon or a cyclic C₃-C₈ hydrocarbon (i.e. acycloalkyl group such as a cyclopropyl group, a cyclobutyl group, acyclopentyl group, a cyclohexyl group or a cyclohexylmethylene group)that can be completely saturated. When used herein, the term “alkyl”refers to a group that may be substituted or unsubstituted. In someembodiments, the term “alkyl” is also intended to refer to thosecompounds wherein one or more methylene groups in the alkyl chain can bereplaced by a heteroatom such as —O—, —Si— or —S—. In some embodiments,alkyl groups can be straight chained or branched C₂-C₆ hydrocarbons orcyclic C₃-C₆ hydrocarbons that can be completely saturated.

x.) As used herein, the term “alkylene” refers to a straight or branchedalkyl chain or a cyclic alkyl group that comprises at least two pointsof attachment to at least two moieties (e.g., —{CH₂}— (methylene),—{CH₂CH₂}—, (ethylene),

etc., wherein the brackets indicate the points of attachment. When usedherein the term “alkylene” refers to a group that may be substituted orunsubstituted. In some embodiments, the term “alkylene” is also intendedto refer to those compounds wherein one or more methylene groups, ifany, in an alkyl chain of the alkylene group can be replaced by aheteroatom such as —O—, —Si— or —S—. In some embodiments, an alkylenegroup can be a C₁-C₁₀ hydrocarbon. In some embodiments, an alkylenegroup can be a C₂-C₆ hydrocarbon.

y.) As used herein, the term “alkenyl” refers to a straight chained orbranched C₂-C₈ hydrocarbon or a cyclic C₃-C₈ hydrocarbon that comprisesone or more double bonds. When used herein, the term “alkenyl” refers toa group that can be substituted or unsubstituted. In some embodiments,the term “alkenyl” is also intended to refer to those compounds whereinone or more methylene groups, if any, in an alkyl chain of the alkenylgroup can be replaced by a heteroatom such as —O—, —Si— or —S—. In someembodiments, alkenyl groups can be straight chained or branched C₂-C₆hydrocarbons or cyclic C₃-C₆ hydrocarbons that comprise one or moredouble bonds.

z.) As used herein, the term “alkenylene” refers to an alkenyl groupthat comprises two points of attachment to at least two moieties. Whenused herein the term “alkenylene” refers to a group that may besubstituted or unsubstituted. In some embodiments, the term “alkenylene”is also intended to refer to those compounds wherein one or moremethylene groups, if any, in an alkyl chain of the alkenylene group canbe replaced by a heteroatom such as —O—, —Si— or —S—.

aa.) As used herein, the term “alkynyl” refers to a straight chained orbranched C₂-C₈ hydrocarbon or a cyclic C₃-C₈ hydrocarbon that comprisesone or more triple bonds.

When used herein, the term “alkynyl” refers to a group that may besubstituted or unsubstituted. In some embodiments, the term “alkynyl” isalso intended to refer to those compounds wherein one or more methylenegroups, if any, in an alkyl chain of the alkynyl group can be replacedby a heteroatom such as —O—, —Si— or —S—. In some embodiments, alkynylgroups can be straight chained or branched C₂-C₆ hydrocarbons or cyclicC₃-C₆ hydrocarbons that have one or more triple bonds.

ab.) As used herein, the term “alkynylene” refers to an alkynyl groupthat comprises two points of attachment to at least two moieties. Whenused herein the term “alkynylene” refers to a group that may besubstituted or unsubstituted. In some embodiments, the term “alkynylene”is also intended to refer to those compounds wherein one or moremethylene groups, if any, in an alkyl chain of the alkynylene group canbe replaced by a heteroatom such as —O—, —Si— or —S—.

ac.) As used herein, the term “aliphatic” refers to any of the straight,branched, or cyclic alkyl, alkenyl, and alkynyl moieties as definedabove. When used herein the term “aliphatic” refers to a group that maybe substituted or unsubstituted.

ad.) As used herein, the term “aryl”, either alone or as part of anothermoiety (e.g., arylalkyl, etc.), refers to carbocyclic aromatic groupssuch as phenyl. Aryl groups also include fused polycyclic aromatic ringsystems in which a carbocyclic aromatic ring is fused to anothercarbocyclic aromatic ring (e.g., 1-naphthyl, 2-naphthyl, 1-anthracyl,2-anthracyl, etc.) or in which a carbocylic aromatic ring is fused toone or more carbocyclic non-aromatic rings (e.g., tetrahydronaphthylene,indan, etc.). As used herein, the term “aryl” refers to a group that maybe substituted or unsubstituted.

ae.) As used herein, the term “heteroaryl,” refers to an aromaticheterocycle that comprises 1, 2, 3 or 4 heteroatoms selected,independently of the others, from nitrogen, sulfur and oxygen. As usedherein, the term “heteroaryl” refers to a group that may be substitutedor unsubstituted. A heteroaryl may be fused to one or two rings, such asa cycloalkyl, an aryl, or a heteroaryl ring. The point of attachment ofa heteroaryl to a molecule may be on the heteroaryl, cycloalkyl,heterocycloalkyl or aryl ring, and the heteroaryl group may be attachedthrough carbon or a heteroatom. Examples of heteroaryl groups includeimidazolyl, furyl, pyrrolyl, thienyl, thiazolyl, isoxazolyl,isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidyl,pyrazinyl, pyridazinyl, quinolyl, isoquinolinyl, indazolyl,benzoxazolyl, benzisooxazolyl, benzofuryl, benzothiazolyl, indolizinyl,imidazopyridinyl, pyrazolyl, triazolyl, oxazolyl, tetrazolyl,benzimidazolyl, benzoisothiazolyl, benzothiadiazolyl, benzoxadiazolyl,indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl,purinyl, pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl orbenzo(b)thienyl, each of which can be optionally substituted.

af.) As used herein, the term “arylene” refers to an aryl or heteroarylgroup that comprises at least two points of attachment to at least twomoieties (e.g., phenylene, etc.). The point of attachment of an arylenefused to a carbocyclic, non-aromatic ring may be on either the aromatic,non-aromatic ring. As used herein, the term “arylene” refers to a groupthat may be substituted or unsubstituted.

ag.) As used herein, the term “arylalkyl” refers to an aryl orheteroaryl group that is attached to another moiety via an alkylenelinker. As used herein, the term “arylalkyl” refers to a group that maybe substituted or unsubstituted. In some embodiments, the term“arylalkyl” is also intended to refer to those compounds wherein one ormore methylene groups, if any, in the alkyl chain of the arylalkyl groupcan be replaced by a heteroatom such as —O—, —Si— or —S—.

ah.) As used herein, the term “arylalkylene” refers to an arylalkylgroup that has at least two points of attachment to at least twomoieties. The second point of attachment can be on either the aromaticring or the alkylene group. As used herein, the term “arylalkylene”refers to a group that may be substituted or unsubstituted. In someembodiments, the term “arylalkylene” is also intended to refer to thosecompounds wherein one or more methylene groups, if any, in the alkylchain of the arylalkylene group can be replaced by a heteroatom such as—O—, —Si— or —S—. When an arylalkylene is substituted, the substituentsmay be on either or both of the aromatic ring or the alkylene portion ofthe arylalkylene.

ai.) As used herein, the terms “optionally substituted” and “substitutedor unsubstituted” are equivalent and interchangeable. Suitablesubstituents for any an alkyl, an alkylene, an alkenyl, an alkenylene,an alkynyl, an alkynylene, an aryl, an aryl alkyl, an arylene, aheteroaryl or an arylalkylene group includes any substituent that isstable under the reaction conditions used in embodiments of thisinvention. Non limiting examples of suitable substituents can include:an alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec butyl, t-butyl, cyclohexyl, etc.) group, a haloalkyl (e.g.,trifluoromethyl, 2,2,2-trifluoroethyl-, etc.) group, an alkoxy (e.g.,methoxy, ethoxy, etc.) group, an aryl (e.g., phenyl) group, an arylalkyl(e.g., benzyl) group, a nitro group (—NO₂), a cyano group (—CN), aquaternized nitrogen atom or a halogen group (e.g., fluorine, chlorine,bromine and/or iodine) group.

In addition, a portion of an alkyl, an alkylene, an alkenyl, analkenylene, an alkynyl, an alkynylene, an aryl, an aryl alkyl, anarylene, a heteroaryl or an arylalkylene group may optionally besubstituted with ═O or ═S.

aj.) As used herein, the term “active ester” refers to compounds thatcan react readily under basic conditions with amines, alcohols andcertain thiols to provide amides, esters and thioesters, respectively.Additional reference is made to: Leo A Paquette, Encyclopedia ofReagents for Organic Synthesis, Vol. 2, John Wiley and Sons, New York,1995 as evidence that active ester is a term well-established in fieldof organic chemistry.

ak.) As used herein, the term “heterocyclic ring” refers to any cyclicmolecular structure comprising atoms of at least two different elementsin the ring or rings. Additional reference is made to: Oxford Dictionaryof Biochemistry and Molecular Biology, Oxford University Press, Oxford,1997 as evidence that heterocyclic ring is a term well-established infield of organic chemistry.

al.) As used herein, the term “leaving group” refers to any atom orgroup, charged or uncharged, that departs during a substitution ordisplacement reaction from what is regarded as the residual or main partof the substrate of the reaction. Additional reference is made to:Oxford Dictionary of Biochemistry and Molecular Biology, OxfordUniversity Press, Oxford, 1997 as evidence that leaving group is a termwell-established in field of organic chemistry.

am.) As used herein, the term “protecting group” refers to a chemicalgroup that is reacted with, and bound to, a functional group in amolecule to prevent the functional group from participating insubsequent reactions of the molecule but which group can subsequently beremoved to thereby regenerate the unprotected functional group.Additional reference is made to: Oxford Dictionary of Biochemistry andMolecular Biology, Oxford University Press, Oxford, 1997 as evidencethat protecting group is a term well-established in field of organicchemistry.

DESCRIPTION OF VARIOUS EMBODIMENTS

It is to be understood that the discussion set forth below in this“General” section can pertain to some, or to all, of the variousembodiments of the invention described herein.

I. General

The Labeling Reagent:

As discussed previously, a labeling reagent that can be used for theanalysis of an analyte or analytes using mass spectrometry can comprisea reporter moiety, a balance moiety (or linker moiety) and a reactivegroup. Novel labeling reagents disclosed herein also comprise at leastone non-encoded detectable label such as a chromophore, a fluorophore, aspin label, an enzyme or a chemiluminescent compound and, in someembodiments, can be represented by the general formula I;

including a salt form and/or a hydrate form thereof. RG is a reactivegroup as described in more detail below. RP is a reporter moiety asdescribed in more detail below. DL is a non-encoded detectable label asdiscussed in more detail below. LK is a linker moiety as described inmore detail below. X is a covalent bond as described in more detailbelow. Y and W are each independently a covalent bond or a linking groupas described in more detail below and m and n are each 0 or 1 providedthat m+n=1.

In some embodiments, the labeling reagent can be support-bound. Thus, insome embodiments, the labeling reagent can be represented by the generalformula I^(ss):

including a salt form and/or a hydrate form thereof. RG is a reactivegroup as described in more detail below. RP is a reporter moiety asdescribed in more detail below. DL is a non-encoded detectable label asdiscussed in more detail below. LK is a linker moiety as described inmore detail below. SS is a solid support to which the reporter moiety“RP” of the labeling reagent is covalently linked through a cleavablelinker as described in more detail below. X is a covalent bond asdescribed in more detail below. Y and W are each independently acovalent bond or a linking group as described in more detail below and mand n are each 0 or 1 provided that m+n=1.The Reactive Group:

The reactive group (sometimes represented by use of the shorthand “RG”)of the labeling reagent or reagents used in various embodiments cancomprise any electrophilic or a nucleophilic group that is capable ofreacting with one or more functional groups of one or more reactiveanalytes of a sample. The reactive group reacts with a functional groupof the analyte to thereby covalently link the analyte to the labelingreagent. Thus, each labeling reagent is capable of reacting with the oneor more reactive analytes of a sample to thereby form the one or morelabeled analytes from the sample.

It is to be understood that in some embodiments, the reactive group maybe considered to include an atom or group associated with the linkermoiety (i.e. balance moiety). For example, if the reactive group is anactive ester or carboxylic acid halide group, the carbonyl group of theactive ester or carboxylic acid halide may, in some embodiments, also beconsidered to be associated with the linker moiety for purposes ofbalancing the mass of the reporter moiety within a set of isomericand/or isobaric labeling reagents where the carbonyl carbon is presentin both the labeling reagent and in the labeled analyte. This willtypically be apparent where heavy atom isotopes are included in thecarbonyl group of some or all of the labeling reagents of a set ofisomeric and/or isobaric labeling reagents. Consequently, in someembodiments, the reactive group can be understood to merely representthe leaving group of a reactive group while in other embodiments thereactive group can comprise additional atoms or moieties that areassociated with the linker moiety. Thus, it is also to be understoodthat when the reactive group is represented by some of the specificmoieties discussed below, the analyte (may be linked to the linkermoiety (i.e. balance moiety) through one or more additional atoms orgroups that may, or may not, be considered to be part of the linkermoiety.

The reactive group can be preexisting or it can be prepared in-situ.In-situ preparation of the reactive group can proceed in the absence ofthe reactive analyte or it can proceed in the presence of the reactiveanalyte. For example, a carboxylic acid group can be modified in-situwith water-soluble carbodiimide (e.g.1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EDC) tothereby prepare an electrophilic group that can be reacted with anucleophile such as an alkyl or aryl amine group of the analyte. In someembodiments, activation of the carboxylic acid group of a labelingreagent with EDC can be performed in the presence of an amine (or othernucleophile) containing analyte. In some embodiments, the amine (orother nucleophile) containing analyte can also be added after theinitial reaction with EDC is performed. In other embodiments, thereactive group can be generated in-situ by the in-situ removal of aprotecting group. Consequently, any existing or newly created reagent orreagents that can effect the derivatization of analytes by the reactionof nucleophiles and/or electrophiles are contemplated by the method,mixture, kit and/or composition embodiments of this invention.

Where the reactive group of the labeling reagent comprises anelectrophile, it can react with a suitable nucleophilic group of theanalyte or analytes. Where the reactive group of the labeling reagentcomprises a nucleophile, it can react with a suitable electrophilicgroup of the analyte or analytes. Numerous pairs of suitablenucleophilic groups and electrophilic groups are known and often used inthe chemical and biochemical arts. Non-limiting examples of reagentscomprising suitable nucleophilic or electrophilic groups that can becoupled to analytes (e.g. such as proteins, peptides, nucleic acids,carbohydrates, lipids, steroids, vitamins, prostaglandins or other smallmolecules having a mass of less that 1500 daltons) to effect theirderivatization, are described in the Pierce Life Science & AnalyticalResearch Products Catalog & Handbook (a Perstorp Biotec Company),Rockford, Ill. 61105, USA. Other suitable reagents are well known in theart and are commercially available from numerous other vendors such asSigma-Aldrich.

The reactive group of a labeling reagent can comprise an amine reactivegroup. For example, the amine reactive group can be an active ester.Active esters are well known in peptide synthesis and refer to certainesters that can be easily reacted with the N-α amine of an amino acidunder conditions commonly used in peptide synthesis. (See: Leo APaquette, Encyclopedia of Reagents for Organic Synthesis, Vol. 2, JohnWiley and Sons, New York, 1995) The amine reactive active ester groupcan be an N-hydroxysuccinimidyl ester (NHS), aN-hydroxysulfosuccinimidyl ester (NHSS), a pentafluorophenyl ester(Pfp), a 2-nitrophenyl ester (2-NP), a 3-nitrophenyl ester (3-NP) a4-nitrophenyl ester (4-NP), a 2,4-dinitrophenylester (2,4-NP), apentafluorophenyl ester (Pfp), a pentachlorophenyl ester (Pcp),3-hydroxy-1,2,3-benzotriazine-4(3H)-one ester (Dhbt),hydroxypyrrolidinone ester (NHP), a 2,4-dihalophenyl ester, a2,2,2-trifluoroethanyl ester or a 1,1,1,3,3,3-hexafluoro-2-propanylester. For example, the leaving group of an active ester (referred toherein generally as Z′ in some embodiments such that in the situationwhere the variable RG is synonymous with only the leaving group portionof the reactive group) can be represented by the following formulas:

wherein X′ is —O— or —S— and each X″ is, independently of the other, —F,—Cl, —Br or —I (See: United States Published Patent Application No. US2005-0148771 A1 for a more detailed description of the synthesis ofactive esters of N-methyl piperazine compounds and FIG. 7 for anillustration of various synthetic routes to said active esters). All ofthe foregoing being alcohol or thiol leaving groups of an active esterwherein said alcohol or thiol leaving group can be displaced by thereaction of the N-α-amine of the amino acid with the carbonyl carbon ofthe active ester group. Accordingly, it should be apparent to theordinary practitioner that the active ester (e.g. N-hydroxysuccinimidylester) of any suitable labelling/tagging reagent described herein couldbe prepared using well-known procedures in combination with thedisclosure provided herein (Also see for example: Greg T. Hermanson(1996). “The Chemistry of Reactive Groups” in “Bioconjugate Techniques”,Chapter 2, pages 137-165, Academic Press, (New York); and also see:“Innovation And Perspectives In Solid Phase Synthesis”, Editor: RogerEpton, SPCC (UK) Ltd, Birmingham, 1990).

In some embodiments, the reactive group of the labelling reagent cancomprise a mixed anhydride since mixed anhydrides are known toefficiently react with amine groups to thereby produce amide bonds.Mixed anhydrides are well known and can be prepared using well-knownmethods often applied in the fields of organic and/or peptide chemistry.

In some embodiments, the reactive group of the labelling reagent can bea carboxylic acid halide group, such as an acid fluoride group (See:Carpino et al., J. Am. Chem. Soc., 112: 9651 (1990)) or acid chloridegroup. Reaction of the acid halide group with an amine of the analytewill form an amide. Reaction of the acid halide group with a hydroxyl orthiol group of the analyte will produce an ester or thio ester,respectively.

In some embodiments, the reactive group can be a sulfonic acid group ofa sulfonyl halide. If a sulfonic acid, it can be activated for reactionwith the analyte. If a sulfonyl halide, it can be reacted directly withthe analyte as this is an activated form.

In some embodiments, the reactive group can be an isocyanate orisothiocyanate group.

In some embodiments, the reactive group of a labeling reagent cancomprise a thiol reactive group. For example, the thiol reactive groupcan be a malemide group, an alkyl halide group, an α-halo-acyl group, anα-halo thione group or an α-halo imine group. By ‘halide group’ or‘halo’ or ‘halo group’ we mean atoms of fluorine, chlorine, bromine oriodine. Said thiol reactive groups are well known and can be preparedusing methods often applied in the field of peptide chemistry.

In some embodiments, the reactive group of a labeling reagent cancomprise a hydroxyl reactive group. For example, the hydroxyl reactivegroup can be a trityl-halide or a silyl-halide reactive moiety. Thetrityl-halide reactive moieties can be substituted (e.g.X′″-methoxytrityl, X′″-dimethoxytrityl, X′″-trimethoxytrityl, etc) orunsubstituted wherein X′″ is the bond that links the reactive group tothe linker (i.e. balance). The silyl reactive moieties can be alkylsubstituted silyl halides, such as X′″-dimethylsilyl,X′″-ditriethylsilyl, X′″-dipropylsilyl, X′″-diisopropylsilyl, etc.)wherein X′″ is the bond that links the reactive group to the linker(i.e. balance). Said reactive groups are well known and can be preparedusing methods often applied in the field of nucleic acid chemistry.

In some embodiments, the reactive group of the labeling reagent comprisea nucleophile such as an amine group, a hydroxyl group, a thiol group ora hydrazide group. In some embodiments, the nucleophilic reactive groupcan be an aminoalkyl group, a hydroxyalkyl group or a thioalkyl group.Said reactive groups are well known and can be prepared using methodsoften applied in the field of organic chemistry.

The Reporter Moiety:

The reporter moiety (sometimes represented by use of the shorthand “RP”)of the labeling reagent or reagents used in embodiments of thisinvention is a group that has a unique mass (or mass to charge ratio ina mass spectrometer) that can be determined. Accordingly, in someembodiments, each reporter moiety of a set of isomeric and/or isobariclabeling reagents has a unique gross mass, and its correspondingsignature ion, is different for each labeling reagent of the set.

Different reporter moieties can comprise one or more heavy atom isotopesto achieve their unique gross mass. For example, isotopes of carbon(¹²C, ¹³C and ¹⁴C), nitrogen (¹⁴N and ¹⁵N), oxygen (¹⁶O and ¹⁸O), sulfur(³²S and ³⁴S) or hydrogen (hydrogen, deuterium and tritium) exist andcan be used in the preparation of a diverse group of reporter moieties.These are not limiting as other light and heavy atom isotopes can alsobe used in the reporter moieties. Basic starting materials suitable forpreparing reporter moieties comprising light and heavy atom isotopes areavailable from various commercial sources such as Cambridge IsotopeLaboratories, Andover, Mass. (See: list of “basic starting materials” atwww.isotope.com) and Isotec (a division of Sigma-Aldrich). CambridgeIsotope Laboratories and Isotec will also prepare desired compoundsunder custom synthesis contracts. Id.

The reporter moiety can either comprise a fixed charge or can becomeionized during the analysis process. Because the reporter moiety caneither comprises a fixed charge or can become ionized, the labelingreagent might be isolated or be used to label the reactive analyte in asalt (or a mixture of salts) or zwitterionic form. Ionization of thereporter moiety (or reporter ion) facilitates its determination in amass spectrometer. Accordingly, the presence of the reporter moiety in alabeled analyte can be determined as a fragment ion, sometimes referredto as a signature ion (or reporter ion). Various reporter ions of anN-methyl piperazine reporter moiety are illustrated in FIG. 5. Themolecular formulas of the ions are ¹³CC₅H₁₃N₂ ⁺, ¹³CC₅H₁₃ ¹⁵NN⁺,¹³C₂C₄H₁₃ ¹⁵NN⁺ and ¹³C₃C₃H₁₃ ¹⁵NN⁺.

When ionized, the signature ion (i.e. reporter ion) can comprise one ormore net positive or negative charges. Thus, the reporter ion cancomprise one or more acidic groups and/or basic groups since such groupscan be easily ionized in a mass spectrometer. For example, the reportermoiety can comprise one or more basic nitrogen atoms (positive charge)and/or one or more ionizable acidic groups such as a carboxylic acidgroup, sulfonic acid group or phosphoric acid group (negative charge)provided that on balance there are more of one or the other of the basicor acidic groups such that the reporter moiety produces a reporter ioncomprising a net positive or negative charge. Non-limiting examples ofreporter moieties comprising at least one basic nitrogen includesubstituted or unsubstituted morpholine, piperidine or piperazinecontaining compounds.

A unique reporter moiety can be associated with a sample of interestthereby labeling one or multiple analytes of that sample with saidunique reporter moiety. In this way information about the uniquereporter moiety (generally detected as a signature ion (i.e. reporterion) in a mass spectrometer) can be associated with information aboutone or all of the analytes of said sample.

However, the unique reporter moiety need not be physically linked to ananalyte when the signature ion is determined. Rather, the unique grossmass of the signature ion can, for example, be determined in a secondmass analysis of a tandem mass analyzer, after ions of the labeledanalyte are fragmented to thereby produce daughter fragment ions andsignature ions.

The determined signature ion can be used to identify the sample fromwhich a determined analyte originated. Further, the amount (oftenexpressed as a concentration and/or quantity) of the unique signatureion, either relative to the amount of other signature ions or relativeto the signature ion associated with a calibration standard (e.g. ananalyte expected in the sample and labeled with a specific reportermoiety), can be used to determine the relative and/or absolute amount(often expressed as a concentration and/or quantity) of analyte in thesample or samples (such as those used to form a sample mixture). In someembodiments, rather than using an internal calibration standard,absolute quantification can be determined based on comparison of thepeak intensities of the various signature ions with a calibration curve.Therefore information, such as the amount of one or more analytes in aparticular sample, can be associated with the reporter moiety that isused to label each particular sample. Where the identity of the analyteor analytes is also determined, that information can be correlated withinformation pertaining to the different signature ions to therebyfacilitate the determination of the identity and amount of each labeledanalyte in one sample or in a plurality of samples.

In some embodiments, the reporter moiety can comprise a nitrogen atomcovalently linked to the methylene carbon of a substituted orunsubstituted N-alkylated acetic acid moiety wherein the substituted orunsubstituted methylene carbon but not the carbonyl group of thecarboxylic acid (or thiocarboxylic acid) group of the acetic acid moietyis part of the reporter. Thus, in some embodiments, the carboxylic acid(or thio carboxylic acid) group can be used to link the reporter to thelinker but it is not considered part of the reporter. The nitrogen atomcan be alkylated with one, two or three groups. For example, the moietycomprising the nitrogen atom can be a substituted primary amine such asa methyl, ethyl or propyl group or a substituted secondary amine such asdimethylamine, diethylamine, di-n-propylamine or diisopropylamine. Thus,for example the reporter moiety “RP” can be illustrated by formulas X¹,X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, or X⁹ as follows wherein the reporter moiety“RP” is set off by the bracket:

The reporter moiety can be a 5, 6 or 7 membered heterocyclic ringcomprising a ring nitrogen atom covalently linked to the methylenecarbon of a substituted or unsubstituted N-alkylated acetic acid moietyto which the analyte is (directly or indirectly) linked through thecarbonyl carbon of the N-alkyl acetic acid moiety and wherein thesubstituted or unsubstituted methylene carbon but not the carbonyl groupof carboxylic acid group is part of the reporter. The heterocyclic ringcan be aromatic or non-aromatic. Thus, the reporter moiety can berepresented by formula Y-J- wherein the group Y can represent the 5, 6or 7 membered heterocyclic ring and the group J can represent thesubstituted or unsubstituted methylene group of the substituted orunsubstituted acetic acid moiety. The heterocyclic ring can besubstituted or unsubstituted. For example, substituents of theheterocylic moiety can include alkyl, alkoxy and/or aryl groups. Thesubstituents can comprise protected or unprotected groups, such asamine, hydroxyl or thiol groups, suitable for linking the analyte to asupport (See FIG. 6). The heterocyclic ring can comprise additionalheteroatoms such as one or more silicon, nitrogen, oxygen and/or sulfuratoms. Thus, for example the reporter moiety “RP” can be illustrated byformulas X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, X¹⁶, X¹⁷, X¹⁸, X¹⁹, X²⁰, X²¹,X²², X²³, X²⁴, X²⁵, or X²⁶ as follows wherein the reporter moiety “RP”is set off by the bracket:

The reporter moiety can be selected so that it does not substantiallysub-fragment under conditions typical for the analysis of the analyte.For the avoidance of any doubt, this is an optional, not a required,feature. For example, the reporter can be chosen so that it does notsubstantially sub-fragment under conditions of dissociative energyapplied to cause fragmentation of the labeled analyte in a massspectrometer. By “does not substantially sub-fragment” we mean thatfragments of the reporter are difficult or impossible to detect abovebackground noise when applied to the successful determination of thelabeled analyte.

In some embodiments, the gross mass of a reporter ion can beintentionally selected to be different as compared with the mass of theanalyte sought to be determined or the mass of any of the expectedfragments of the analyte (i.e. a “quiet zone”). For example, whereproteins or peptides are the analytes, the gross mass of the reporterion can be chosen to be different as compared with any naturallyoccurring amino acid or peptide, or expected fragment ion thereof. Thiscan facilitate analyte determination since, depending on the analyte,the lack of any possible components of the sample having the samecoincident mass can add confidence to the result of any analysis.Examples of mass ranges where little background can be expected forpeptides can be found in Table 1.

TABLE 1 Possible “Quiet Zones” For Selection Of Label Fragment Ion m/zAssociated With Peptide Analysis M/z start-end 10-14 19-22 24-26 31-3840-40 46-50 52-52 58-58 61-69 71-71 74-83 89-97 103-109 113-119 121-125128-128 131-135 137-147 149-154 156-156 160-174 177-182 184-184 188-189191-191 202-207 210-210 216-222 224-226

The reporter moiety can be non-polymeric. The reporter moiety can beselected to produce a signature ion of m/z that indicates its mass isless than 250 atomic mass units (amu). The reporter moiety can beselected to produce a signature ion of m/z less than 200 amu. Thereporter moiety can be selected to produce a signature ion of m/z lessthan 150 amu. Such a small molecule can be easily determined in thesecond mass analysis, free from other components of the sample havingthe same coincident mass in the first mass analysis. In this context,the second mass analysis can be performed, typically in a tandem massspectrometer (or, for example by post source decay in a single stageinstrument), on selected ions that are determined in the first massanalysis. Because ions of a particular mass to charge ratio can bespecifically selected out of the first mass analysis for possiblefragmentation and further mass analysis, the non-selected ions from thefirst mass analysis are not carried forward to the second mass analysisand therefore do not contaminate the spectrum of the second massanalysis. Furthermore, the sensitivity of a mass spectrometer and thelinearity of the detector (for purposes of quantification) can be quiterobust in this low mass range. Additionally, the present state of massspectrometer technology can allow for baseline mass resolution of lessthan one Dalton in this mass range. For all these reasons, reporterspossessing the above described characteristics can provide quiteaccurate quantification of determined analytes from complex mixturesutilizing methods as described herein.

The Linker (or Balance) Moiety:

The linker (or balance) moiety (sometimes referred to by use of theshorthand “LK”) of the labeling reagent or reagents can be used withembodiments of this invention to link the reporter moiety (RP) and thenon-encoded detectable label (DL) to the analyte or the reactive groupdepending on whether or not a reaction with the analyte has occurred.The linker moiety can be branched or unbranched. For example, the linkercan be branched where the reporter and the non-encoded detectable labeleach, independently of the other, are linked to the linker but may belinear where the non-encoded detectable label and reporter moiety arelinked in sequence to the linker moiety (c.f. FIGS. 1 a and 1 b). Thenon-encoded detectable label may be linked directly or indirectly to thelinker.

The linker can be selected to produce a neutral species (i.e. undergoneutral loss in a mass spectrometer) wherein both the bond that linksthe linker to the reporter moiety (i.e. the X bond) and the bond orgroup that links the linker to the analyte (in some embodiments this isY) fragment in a mass spectrometer. In some embodiments, the linkermoiety can be designed to sub-fragment when subjected to dissociativeenergy, including sub-fragmentation to thereby produce only neutralfragments of the linker. In some embodiments, the linker can be designedto produce one or more detectable fragments.

When used in a set and/or kit comprising a set of isomeric and/orisobaric labeling reagents, the linker moiety can comprise one or moreheavy atom isotopes such that the mass of the linker moiety of eachdifferent reagent of the set compensates for the difference in grossmass between the reporter moieties for the different labeling reagentsof the set such that the aggregate gross mass of the combination of thereporter moiety and the linker moiety is the same for each labelingreagent of the set. Thus, the aggregate gross mass (i.e. the gross masstaken as a whole) of the reporter/linker combination (i.e. thereporter/linker moiety) can be the same (on a gross basis) for eachlabeled analyte of a mixture or for the labeling reagents of set and/orkit. More specifically, the linker moiety can compensate for thedifference in gross mass between reporter moieties of labeled analytesfrom different samples wherein the unique gross mass of the reportermoiety correlates with the sample from which the labeled analyteoriginated and the aggregate gross mass of the reporter/linkercombination is the same for each labeled analyte of a sample mixtureregardless of the sample from which it originated. In this way, thegross mass of identical analytes in two or more different samples canhave the same gross mass when labeled and then mixed to produce a samplemixture.

For example, the labeled analytes, or the labeling reagents of a setand/or kit for labeling the analytes, can be isomers and/or isobars.Thus, if ions of a particular mass to charge ratio (taken from thesample mixture) are selected (i.e. selected ions) in a mass spectrometerfrom a first mass analysis of the sample mixture, identical analytesfrom the different samples that make up the sample mixture can berepresented in the selected ions in proportion to their respectiveconcentration and/or quantity in the sample mixture since they all havethe same gross mass regardless of which label is linked to the analyte.Accordingly, the linker not only links the reporter and non-encodeddetectable label to the analyte, it also serves to compensate for thediffering masses of the unique reporter moieties to thereby harmonizethe gross mass of the reporter/linker moiety in the labeled analytes ofthe various samples.

Because the linker can act as a mass balance for the reporter moietiesin the labeling reagents, the greater the number of atoms in the linker,the greater the possible number of different isomeric/isobaric labelingreagents of a set and/or kit. Stated differently, generally the greaterthe number of atoms that a linker comprises, the greater the number ofpotential reporter/linker combinations since isotopes can be substitutedat most any position in the linker to thereby produce isomers and/orisobars of the linker portion wherein the linker portion is used tooffset the differing masses of the reporter portion and thereby create aset of unique isomeric and/or isobaric labeling reagents. Such diversesets of labeling reagents are particularly well suited for multiplexanalysis of analytes in the same and/or different samples.

The total number of labeling reagents of a set and/or kit can be two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty or more. Based upon mass balancingconsiderations, the diversity of the labeling reagents of a set or kitis limited only by the number of atoms of the reporter and linkermoieties, the heavy atom isotopes available to substitute for the lightisotopes and the various synthetic configurations in which the isotopescan be synthetically placed. As suggested above however, numerousisotopically enriched basic starting materials are readily availablefrom manufacturers such as Cambridge Isotope Laboratories and Isotec.Such isotopically enriched basic starting materials can be used by theordinary practitioner in synthetic processes used to produce sets ofisobaric and/or isomeric labeling reagents or be used to produce theisotopically enriched starting materials (i.e. synthetically enrichedcompounds) that can be used in the synthetic processes used to producesets of isobaric and/or isomeric labeling reagents.

The Reporter/Linker Combination (i.e. the Reporter/Linker Moiety):

The labeling reagents can comprise reporter moieties and linker moietiesthat are linked directly to each other through the bond X. As describedabove, the reporter/linker moiety can be identical in gross mass foreach member of a set and/or kit of labeling reagents or for each labeledanalyte in a mixture of labeled analytes. Moreover, the bond (i.e. bondX) that links the reporter moiety (directly or indirectly) to the linkermoiety can be designed to fragment, in at least a portion of theselected ions, when subjected to dissociative energy thereby releasingthe signature ion (i.e. the reporter ion) from the linker moiety,linker/analyte and/or linker/analyte/non-encoded detectable labelmoiety. Accordingly, the gross mass of the signature ion (observed as amass to charge (m/z) ratio in the mass spectrometer) and its intensity(i.e. its peak intensity) can be observed directly in MS/MS analysis.

The reporter/linker moiety can comprise various combinations of the sameor different heavy atom isotopes amongst the various labeling reagentsof a set or kit. In the scientific literature this has sometimes beenreferred to as “coding”, “isotope coding” or simply as “encoding”. Forexample, Abersold et al. has disclosed the isotope coded affinity tag(ICAT; see WO00/11208). In one respect, the reagents of Abersold et al.differ from the labeling reagents described herein that Abersold doesnot teach two or more same mass labeling reagents such as isomericand/or isobaric labeling reagents. Rather, Abersold et al. teach about“light” and “heavy” versions of their labeling reagents. Versions of theICAT reagent that comprise a non-encoded detectable label are (sometimesreferred to as VICAT) described in WO 2004/019000, in Lu et al., Anal.Chem., 76: 4104-4111 (2004) and in Bottari et al, BioconjugateChemistry, 15(2): 380-388 (2004).

In some embodiments, the reporter and/or linker moieties can comprise anatom or group that can be used to immobilize the labeling reagent orlabeled analyte to a support. Immobilization can be direct or indirect.For example, direct immobilization can occur if an atom or group (e.g.an alkyl amine substituent of the reporter) associated with the reporterinteracts directly with a reactive group (e.g. the reactive group of acleavable linker) of the support to effect mobilization. By comparison,indirect immobilization occurs if, for example, a substituent of thereporter (e.g. an alkylamine substituent of the reporter and/or linker)is modified (e.g. is biotinylated) and the modifying group interactswith a reactive group of the support (e.g. avidin or streptavidin) toeffect immobilization. Consequently, this invention contemplatesembodiments wherein the analytes can be reacted with support-boundlabeling reagents wherein each support comprises a unique labelingreagent such that different samples are reacted with different supportsas well as embodiments where each different sample is reacted with adifferent labeling reagent and the reaction products are thereafterimmobilized to the same or to different supports. In either case, asample mixture is generally obtained by cleaving the labeled analytesfrom the support(s) for analysis by mass spectrometry (See: FIG. 6). Thereleased labeled analytes of each sample can be optionally collectedseparately or they can be mixed during the cleavage process to therebyform a sample mixture. If collected, the released labeled analytes canthereafter be mixed to form a sample mixture.

The Non-Encoded Detectable Label:

The labeling reagents described herein comprise a non-encoded detectablelabel (sometimes referred to by use of the shorthand “DL”). Non-limitingexamples of non-encoded detectable labels (moieties) that can be usedwith the labeling reagents disclosed herein include, but are not limitedto, a chromophore, a fluorophore, a spin label, an enzyme or achemiluminescent compound. By “non-encoded” we mean that the detectablelabel isn't synthetically enriched with one or more heavy atom isotopes.Accordingly, the non-encoded detectable label doesn't produce a reporterion (signature ion) and doesn't represent part of the linker (balance)moiety.

Non-limiting examples of fluorophores include 5(6)-carboxyfluorescein(Flu), 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid (Cou),5(and 6)-carboxy-X-rhodamine (Rox), Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3)Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5)Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine dyes 2, 3, 3.5, 5and 5.5 are available as NHS esters from Amersham, Arlington Heights,Ill.) or the Alexa dye series (Molecular Probes, Eugene, Oreg.).

Non-limiting examples of enzymes include polymerases (e.g. Taqpolymerase, Klenow DNA polymerase, T7 DNA polymerase, Sequenase, DNApolymerase 1 and phi29 polymerase), alkaline phosphatase (AP),horseradish peroxidase (HRP), soy bean peroxidase (SBP)), ribonucleaseand protease.

For sets of isomeric and/or isobaric labeling reagents, each non-encodeddetectable label has the same gross mass and same net charge as theother non-encoded detectable labels of the labeling reagents of the setunder similar conditions. Each different non-encoded detectable label isindependently detectable of the others in the set. For example, thefluorophores (i.e. the non encoded detectable labels) of the labelingreagents illustrated in FIGS. 8 a and 8 b are believed to havedistinguishable fluorescent profiles, identical net charges atphysiological pH and have the same nominal gross mass.

Because each non-encoded detectable label is independently detectablewithin a set, in some embodiments, the non-encoded detectable labels canbe used for determining where there appears to be a difference inrelative concentrations of a particular labeled analyte (or othercondition of interest) during the electrophoretic separation asdescribed below. Thus, in some embodiments, analytes of interest can bejudiciously selected for MS analysis (and to some degree quantified)based upon an (electrophoretic separation and) analysis that precedesthe MS analysis and quantification. In this way, MS^(n) analysis ofanalytes that don't seem to have changed in amount, or don't seem to bedifferent in relative quantification, with reference to an expectedamount, can be skipped thereby permitting more efficient use of time andresources.

The Bond X:

X is a bond between an atom of the reporter and an atom of the linkermoiety or non-encoded detectable label. Bond X of the various labelingreagents disclosed herein can fragment, in at least a portion ofselected ions, when subjected to dissociative energy. Therefore, thedissociative energy level can be adjusted in a mass spectrometer so thatbond X can fragment in at least a portion of the selected ions of thelabeled analytes. Fragmentation of bond X releases the reporter from theanalyte so that the reporter (i.e. signature ion) can be determinedindependently from ions of the analyte.

The Bond or Linker Group Y:

Y is a bond or linking group that links the linker moiety (“LK”) to thereactive group (“RG”). Whether a bond or a linking group, Y isoptionally fragmentable by application of dissociative energy in a massspectrometer. If Y is a linking group, it can be an alkylene group, analkenylene group, an alkynylene group, an arylene group, or anarylalkylene group. If Y is a linking group, it may optionally compriseone or more heavy atom isotopes. If Y is a linking group and thelabeling reagent is used in a set of isomeric and/or isobaric labelingreagents, its gross mass is the same for every labeling reagent of theset.

If Y is a fragmentable, fragmentation of Y releases thereporter/linker/non-encoded detectable label moiety from the analyte, ora fragment thereof depending on whether or not bond X has already beenfragmented. If a bond, bond Y can be more labile than bond X. Bond X canbe more labile than bond Y. Bonds X and Y can be of the same relativelability.

When the analyte of interest is a protein or peptide, the relativelability of bonds X and Y can be adjusted with regard to an amide(peptide) bond. Bond X, bond Y or both bonds X and Y can be more, equalor less labile as compared with a typical amide (peptide) bond. Forexample, under conditions of dissociative energy, bond X and/or bond Ycan be less prone to fragmentation as compared with the peptide bond ofa Z′″-pro dimer or Z′″-asp dimer, wherein Z′″ is any natural amino acid,pro is proline and asp is aspartic acid. In some embodiments, bonds Xand Y will fragment with approximately the same level of dissociativeenergy as a typical amide bond. In some embodiments, bonds X and Y willfragment at a greater level of dissociative energy as compared with atypical amide bond.

Bonds X and Y can also exist such that fragmentation of bond Y resultsin the fragmentation of bond X, and vice versa. In this way, both bondsX and Y can fragment essentially simultaneously such that no substantialamount of analyte, or daughter fragment ion thereof, comprises a partiallabel in the second mass analysis. By “substantial amount of analyte” wemean that less than 25%, and preferably less than 10%, of partiallylabeled analyte can be determined in the MS/MS spectrum.

Because in some embodiments there can be a clear demarcation betweenlabeled and unlabeled fragments of the analyte in the spectra of thesecond mass analysis (MS/MS), this feature can simplify theidentification of the analytes from computer assisted analysis of thedaughter fragment ion spectra. Moreover, because the fragment ions ofanalytes can, in some embodiments, be either fully labeled or unlabeled(but not partially labeled) with the reporter/linker/detectable moiety,there can be little or no scatter in the masses of the daughter fragmentions caused by isotopic distribution across fractured bonds such aswould be the case where isotopes were present on each side of a singlelabile bond of a partially labeled analyte routinely determined in thesecond mass analysis.

The Bond or Linker Group W:

W is a bond or linking group that links the linker moiety (“LK”) to thenon-encoded detectable label (“DL”). W is optionally cleavable byapplication of light, heat or chemical reagent(s) and/or fragmentable(i.e. cleavable) by application of dissociative energy in a massspectrometer. If W is a linking group, it can be an alkylene group, analkenylene group, an alkynylene group, an arylene group or anarylalkylene group. If W is a linking group, it may optionally compriseone or more heavy atom isotopes. If W is a linking group and thelabeling reagent is used in a set of isomeric and/or isobaric labelingreagents, its gross mass is the same for every labeling reagent of theset. In some embodiments, where W is a linking group, W can comprise,for example, a cleavable moiety such as XAL, PAL or HMPB as described inmore detail in: Kates et al., Solid Phase Synthesis: A Practical Guide,Marcel Dekker, Inc., New York, 2000.

Support-Bound Labeling Reagents Comprising Cleavable Linkers:

According to some embodiments, the analytes from a sample can be reactedwith a solid support comprising a labeling reagent (each sample beingreacted with a different solid support and therefore labeled analytes ofeach sample comprises a different reporter moiety) and the components ofthe sample that do not react with the reactive group can be optionallywashed away. The labeled analyte or analytes can then be removed fromeach solid support by treating the support under conditions that cleavea cleavable linker and thereby release the reporter-linker-non-encodingdetectable label/analyte complex from the support. Each support can besimilarly treated under conditions that cleave the cleavable linker tothereby obtain two or more different samples, each sample comprising oneor more labeled analytes wherein the labeled analytes associated with aparticular sample can be identified and/or quantified by the uniquereporter moiety linked thereto. The collected samples can then be mixedto form a sample mixture, as described below.

Various supports are commercially available that can be used to attach alabeling reagent to the support through a cleavable linker. In someembodiments, the labeling reagent can be attached to the support throughthe reporter moiety. Generally, the labeling reagent will comprise anucleophilic or electrophilic group that can react with a functionalgroup of the cleavable linker to thereby cleavably link the labelingreagent to the support. In some embodiments, the labeling reagent can besynthesized on the support bearing the cleavable linker. In still otherembodiments, the labeling reagent comprising the cleavable linker can bereacted with the support to thereby form the support-bound labelingreagent comprising the cleavable linker.

For example, the amino, hydroxyl or thiol group of a reporter moiety ofa labeling reagent can be reacted with the cleavable linker of asuitable support. The cleavable linker can be a “sterically hinderedcleavable linker”. Cleavage of the cleavable linker will release thelabel or a labeled analyte from the support. Non-limiting examples ofsterically hindered solid supports include: Trityl chloride resin(trityl-Cl, Novabiochem, P/N 01-64-0074), 2-Chlorotrityl chloride resin(Novabiochem, P/N 01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (AppliedBiosystems P/N 400377), 4-methyltrityl chloride resin (Novabiochem, P/N01-64-0075), 4-methoxytrityl chloride resin (Novabiochem, P/N01-64-0076), Hydroxy-(2-chorophnyl)methyl-PS (Novabiochem, P/N01-64-0345), Rink Acid Resin (Novabiochem P/Ns 01-64-0380, 01-64-0202),NovaSyn TGT alcohol resin (Novabiochem, P/N 01-64-0074). Numerous othercleavable linkers are known in the art and can be used to preparesuitable supports using no more than commercially available materials,routine experimentation and the teachings provided herein.

For example, the reporter moiety can be a 5, 6 or 7 member heterocyclicring comprising an atom or group that facilitates the cleavable linkageof it to a suitable support. For example, the group can be an alkylene,alkenylene, alkynylene, arylene or alkylarylene group comprising anamino, hydroxyl or thiol group. In some embodiments, the heterocyclicring doesn't require an additional functional group. For example, theatom that is bound to the cleavable linker can be the secondary nitrogenof a piperazine ring. A discussion of exemplary piperazine compounds andmethods for their manufacture can be found in published United StatesPatent Application No: US 2004-0219685 A1. Reaction of the amino,hydroxyl or thiol group of the reporter moiety with the supportcomprising the linker moiety can form the support comprising thelabeling reagent. Reaction of the support with the support-boundlabeling reagent can produce the support-bound labeled analyte. Sincethe cleavable linker can be selected to be cleavable by light, heat orchemical reagent(s), appropriate treatment will release the labeledanalyte(s) that can be used to produce a sample mixture as describedbelow.

With reference to FIG. 6, an exemplary labeling reagent cleavably linkedto a support is illustrated. As illustrated, a trityl group linked tothe beaded support is the cleavable linker (e.g. Trityl chloride resin(trityl-Cl, Novabiochem, P/N 01-64-0074). As illustrated, the linkedfunctional group Q of an alkylated piperazine reporter moiety of thelabeling reagent is cleavably linked to the support-bound trityl groupand the leaving group Z′ of the reactive group can be displaced by thefunctional group of the analyte to thereby form the support-boundlabeled analyte. For example, the reactive group of the labeling reagentcan be a carboxylic acid that is activated in-situ for reaction with anamine functional group of the analyte (e.g. a peptide) to thereby formthe support-bound labeled analyte. As illustrated, treatment of thesupport with acid releases the labeled analyte from the support byregeneration of the functional group Q′ of the reporter moiety of thelabeling reagent.

Mass Spectrometers/Mass Spectrometry (MS):

The methods of this invention can be practiced using tandem massspectrometers and other mass spectrometers that have the ability toselect and fragment molecular ions. Tandem mass spectrometers (and to alesser degree single-stage mass spectrometers) have the ability toselect and fragment molecular ions according to their mass-to-charge(m/z) ratio, and then record the resulting fragment (daughter) ionspectra. More specifically, daughter fragment ion spectra can begenerated by subjecting selected ions to dissociative energy (e.g.collision-induced dissociation (CID)). For example, ions correspondingto labeled peptides of a particular m/z ratio can be selected from afirst mass analysis, fragmented and reanalyzed in a second massanalysis. Representative instruments that can perform such tandem massanalysis include, but are not limited to, magnetic four-sector, tandemtime-of-flight, triple quadrupole, ion-trap, and hybrid quadrupoletime-of-flight (Q-TOF) mass spectrometers.

These types of mass spectrometers may be used in conjunction with avariety of ionization sources, including, but not limited to,electrospray ionization (ESI) and matrix-assisted laser desorptionionization (MALDI). Ionization sources can be used to generate chargedspecies for the first mass analysis where the analytes do not alreadypossess a fixed charge. Additional mass spectrometry instruments andfragmentation methods include post-source decay in MALDI-MS instrumentsand high-energy CID using MALDI-TOF (time of flight)-TOF MS. For arecent review of tandem mass spectrometers please see: R. Aebersold andD. Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269-295(2001).

Fragmentation By Dissociative Energy:

It is well accepted that bonds can fragment as a result of the processesoccurring in a mass spectrometer. Moreover, bond fragmentation can beinduced in a mass spectrometer by subjecting ions to dissociativeenergy. For example, the dissociative energy can be produced in a massspectrometer by collision-induced dissociation (CID). Other non-limitingexamples of dissociative energy that can be used to fragment ions in amass spectrometer include, but are not limited to, collision activateddissociation (CAD), photoinduced dissociation (PID)), surface induceddissociation (SID)), electron induced dissociation (EID), electroncapture dissociation (ECD)), thermal/black body infrared radiativedissociation (BIRD), post source decay, or combinations thereof. Thoseof ordinary skill in the art of mass spectrometry will appreciate thatother exemplary techniques for imposing dissociative energy that causefragmentation include, but are not limited to, photo dissociation,electron capture and surface induced dissociation.

The process of fragmenting bonds by collision-induced dissociationinvolves increasing the kinetic energy state of selected ions, throughcollision with an inert gas, to a point where bond fragmentation occurs.For example, kinetic energy can be transferred by collision with aninert gas (such as nitrogen, helium or argon) in a collision cell. Theamount of kinetic energy that can be transferred to the ions isproportional to the number of gas molecules that are allowed to enterthe collision cell. When more gas molecules are present, a greateramount of kinetic energy can be transferred to the selected ions, andless kinetic energy is transferred when there are fewer gas moleculespresent.

It is therefore clear that the application of dissociative energy in amass spectrometer can be controlled. It is also well accepted thatcertain bonds are more labile than other bonds. The lability of thebonds in an analyte or the reporter/linker/non-encoded detectable labelmoiety depends upon the nature of the analyte and the nature of thereporter/linker/non-encoded detectable label moiety. Accordingly, thedissociative energy can be adjusted so that the analytes and/or thelabeling reagents (e.g. the reporter/linker combinations) can befragmented in a manner that is determinable. One of skill in the artwill appreciate how to make such routine adjustments to the componentsof a mass spectrometer to thereby achieve the appropriate level ofdissociative energy to thereby fragment at least a portion of ions oflabeled analytes into signature ions (i.e. reporter ions) and daughterfragment ions.

For example, dissociative energy can be applied to ions that areselected/isolated from the first mass analysis. In a tandem massspectrometer, the extracted ions can be subjected to dissociativeenergy, to thereby cause fragmentation, and then transferred to a secondmass analyzer. The selected ions can have a selected mass to chargeratio. The mass to charge ratio can be within a range of mass to chargeratios depending upon the characteristics of the mass spectrometer. Whencollision induced dissociation is used, the ions can be transferred fromthe first to the second mass analyzer by passing them through acollision cell where the dissociative energy can be applied to therebyproduce fragment ions. For example the ions sent to the second massanalyzer for analysis can include some, or a portion, of the remaining(unfragmented) selected ions (if any), as well as reporter ions(signature ions) and daughter fragment ions of the labeled analyte.

Analyte Determination By Computer Assisted Database Analysis:

In some embodiments, analytes can be determined based upon daughter-ionfragmentation patterns that are analyzed by computer-assisted comparisonwith the spectra of known or “theoretical” analytes. For example, thedaughter fragment ion spectrum of a peptide ion fragmented underconditions of low energy CID can be considered the sum of many discretefragmentation events. The common nomenclature differentiates daughterfragment ions according to the amide bond that breaks and the peptidefragment that retains charge following bond fission (Reopstorff et al.,Biomed. Mass Spectrom., 11: 601 (1988)). Charge-retention on theN-terminal side of the fissile amide bond results in the formation of ab-type ion. If the charge remains on the C-terminal side of the brokenamide bond, then the fragment ion is referred to as a y-type ion. Inaddition to b- and y-type ions, the CID mass spectrum may contain otherdiagnostic fragment ions (these include ions generated by neutral lossof ammonia (−17 amu) from glutamine, lysine and arginine or the loss ofwater (−18 amu) from hydroxyl-containing amino acids such as serine andthreonine); the diagnostic fragment ions as well as the b- and y-typeions all being daughter fragment ions. Certain amino acids have beenobserved to fragment more readily under conditions of low-energy CIDthan others. This is particularly apparent for peptides containingproline or aspartic acid residues, and even more so at aspartyl-prolinebonds (Mak, M. et al., Rapid Commun. Mass Spectrom., 12: 837-842(1998)). Accordingly, the peptide bond of a Z′″-pro dimer or Z′″-aspdimer, wherein Z′″ is any natural amino acid, pro is proline and asp isaspartic acid, will tend to be more labile as compared with the peptidebond between all other amino acid dimer combinations.

For peptide and protein samples therefore, low-energy CID spectracontain redundant sequence-specific information in overlapping b- andy-series ions, internal fragment ions from the same peptide, andimmonium and other neutral-loss ions. Interpreting such CID spectra toassemble the amino acid sequence of the parent peptide de novo ischallenging and time-consuming but can be done. Recent advances incomputer assisted de novo methods for sequencing are were described inHuang, Y., Ross, P, Smirnov, I, Martin, S. and Pappin, D. 2003,Proceedings of 6th International Symposium on MS in Health and LifeSciences, Aug. 24-28, 2003, San Francisco Calif. The most significantadvances in identifying peptide sequences have been the development ofcomputer algorithms that correlate peptide CID spectra with peptidesequences that already exist in protein and DNA sequence databases. Suchapproaches are exemplified by programs such as SEQUEST (Eng, J. et al.J. Am. Soc. Mass Spectrom., 5: 976-989 (1994)) and MASCOT (Perkins, D.et al. Electrophoresis, 20: 3551-3567 (1999)).

In brief, experimental peptide CID spectra (MS/MS spectra) are matchedor correlated with ‘theoretical’ daughter fragment ion spectracomputationally generated from peptide sequences obtained from proteinor genome sequence databases. The match or correlation is based upon thesimilarities between the expected mass and the observed mass of thedaughter fragment ions in MS/MS mode. The potential match or correlationis scored according to how well the experimental and ‘theoretical’fragment patterns coincide. The constraints on databases searching for agiven peptide amino acid sequence are so discriminating that a singlepeptide CID spectrum can be adequate for identifying any given proteinin a whole-genome or expressed sequence tag (EST) database. For otherreviews please see: Yates, J. R. Trends, Genetics, 16: 5-8 (2000) andYates, J. R., Electrophoresis 19: 893-900 (1998).

Accordingly, daughter fragment ion analysis of MS/MS spectra can be usednot only to determine the analyte of a labeled analyte, it can also beused to determine analytes from which the determined analyte originated.For example, identification of a peptide in the MS/MS analysis can becan be used to determine the protein from which the peptide was cleavedas a consequence of an enzymatic digestion of the protein. It isenvisioned that such analysis can be applied to other analytes, such asnucleic acids, lipids steroids and/or prostaglandins.

The X Bond and the Y Bond:

The bond between an atom of the reporter moiety and an atom of thelinker moiety is the X bond. If a bond in a labeled analyte, the bondbetween an atom of the linker moiety and an atom of the analyte is the Ybond. In some embodiments, the X bond and the Y bond can fragment, in atleast a portion of selected ions, when subjected to dissociative energy.Therefore, the dissociative energy can, in some embodiments, be adjustedin a mass spectrometer so that both the X bond and the Y bond fragmentin at least a portion of the selected ions of the labeled analytes.

Fragmentation of the X bond releases the reporter moiety from theanalyte so that the reporter ion can be determined independently fromthe analyte. Fragmentation of the Y bond releases thereporter/linker/non-encoded detectable label moiety from the analyte, orthe linker from the analyte, depending on whether or not the Y bond hasalready fragmented. In some embodiments, the X bond can be more labilethan the Y bond. In some embodiments, the Y bond can be more labile thanthe X bond. In some embodiments, the X and Y bonds can be of the samerelative lability. Stated briefly, the X bond is designed to fragment tothereby release the reporter ion but the Y bond may, or may not,fragment in the various embodiments of this invention. If the labelingreagent doesn't comprise a fragmentable Y bond, daughter ion analysiscan be adjusted so that the mass of any modification of analyte by thelabeling reagent is compensated for in relevant analyses.

In some embodiments, when the analyte of interest is a protein orpeptide, the relative lability of the X and Y bonds can be adjusted withregard to an amide (peptide) bond. The X bond, the Y bond or both bondsX and Y can be more, equal or less labile as compared with a typicalamide (peptide) bond. For example, under conditions of dissociativeenergy, the X bond and/or the Y bond can be less prone to fragmentationas compared with the peptide bond of a Z′″-pro dimer or Z′″-asp dimer,wherein Z′″ is any natural amino acid, pro is proline and asp isaspartic acid. In some embodiments, the X bond and the Y bond canfragment with approximately the same level of dissociative energy as atypical amide bond. In some embodiments, the X and Y bonds can fragmentat a greater level of dissociative energy as compared with a typicalamide bond.

In some embodiments, the X bond and the Y bond can exist such thatfragmentation of the X bond results in the fragmentation of the Y bond,and vice versa. In this way, both bonds X and Y can fragment essentiallysimultaneously such that no substantial amount of analyte, or daughterfragment ion thereof, comprises a partial label. By “substantial amountof analyte” we mean that less than 25%, and preferably less than 10%, ofpartially labeled analyte can be determined in the mass spectrometer(e.g. in MS/MS or MS^(n′) analysis, wherein n′ is an integer greaterthan 1).

Because in some embodiments there can be a clear demarcation betweenlabeled and unlabeled fragments of the analyte in the mass spectra (e.g.in MS/MS analysis), this feature can simplify the identification of theanalytes from computer assisted analysis of the daughter fragment ionspectra since no compensation for the remnants of the label need beapplied to the mass calculations used to analyze the daughter fragmentions of an analyte. Moreover, because the fragment ions of analytes can,in some embodiments, be either fully labeled or unlabeled (but notpartially labeled), there can be little or no scatter in the masses ofthe daughter fragment ions caused by isotopic distribution acrossfractured bonds such as would be the case where isotopes were present oneach side of a single labile bond of a partially labeled analyteresulting from fragmentation of the labeled analyte caused by theapplication of dissociative energy levels.

The Labeling of Analytes:

As discussed previously, analytes can be labeled by reacting afunctional group of the analyte with the reactive group of the labelingreagent. The functional group on the analyte can be one of anelectrophilic group or a nucleophilic group and the functional group ofthe labeling reagent can be the other of the electrophilic group or thenucleophilic group. The electrophile and nucleophile can react to form acovalent link between the analyte and the labeling reagent.

The labeling reaction can take place in solution. In some embodiments,one of the analyte or the labeling reagent can be support-bound. Thelabeling reaction can sometimes be performed in aqueous conditions.Aqueous conditions can be selected for the labeling of biomolecules suchas proteins, peptides and/or nucleic acids. The labeling reaction cansometimes be performed in organic solvent or a mixture of organicsolvents. Organic solvents can be selected for analytes that are smallmolecules. Mixtures of water and organic solvent or organic solvents canbe used across a broad range. For example, a solution of water and fromabout 5 percent to about 95 percent organic solvent or solvents (v/v)can be prepared and used for labeling the analyte. In some embodiments,a solution of water and from about 50 percent to about 95 percentorganic solvent or solvents (v/v) can be prepared and used for labelingthe analyte. In some embodiments, a solution of water and from about 65percent to about 80 percent organic solvent or solvents (v/v) can beprepared and used for labeling the analyte. Non-limiting examples oforganic solvents include N,N′-dimethylformamide (DMF), acetonitrile(ACN), N-Methyl pyrrolidine (NMP) and alcohols such as methanol,ethanol, propanol and/or butanol. Those of skill in the art will be ableto determine appropriate solvent conditions to facilitate analytelabeling depending upon the nature of the labeling reagent and thenature of the analyte using no more than knowledge available in the artand the disclosure provided herein in combination with routineexperimentation.

When performing a labeling reaction, the pH can be modulated. The pH canbe in the range of 4-10. The pH can be outside this range. Generally,the basicity of non-aqueous reactions can be modulated by the additionof non-nucleophilic organic bases. Non-limiting examples of suitablebases include N-methylmorpholine, triethylamine andN,N-diisopropylethylamine. Alternatively, the pH of water containingsolvents can be modulated using biological buffers such as(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid) (HEPES) or4-morpholineethane-sulfonic acid (MES) or inorganic buffers such assodium carbonate and/or sodium bicarbonate. Because at least one of thereactive groups can be electrophilic, it can be desirable to select thebuffer to not contain any nucleophilic groups. Those of skill in the artwill, with the application of ordinary experimentation, be able toidentify other buffers that can be used to modulate the pH of a labelingreaction so as to facilitate the labeling of an analyte with a labelingreagent. Accordingly, those of skill in the art will be able todetermine appropriate conditions of solvent and pH to thereby facilitateanalyte labeling depending upon the nature of the labeling reagent andthe nature of the analyte using no more than the disclosure providedherein in combination with routine experimentation.

Sample Processing:

In certain embodiments of this invention, a sample can be processedprior to, as well as after, labeling of the analyte or analytes.Processing can facilitate the labeling of the analyte or analytes. Theprocessing can facilitate the analysis of the sample components.Processing can simplify the handling of the samples. Processing canfacilitate two or more of the foregoing.

For example, a sample can be treated with an enzyme or a chemical. Theenzyme can be a protease (to degrade proteins and peptides), a nuclease(to degrade nucleic acids) or some other enzyme. The enzyme can bechosen to have a very predictable degradation pattern. Two or moreproteases and/or two or more nuclease enzymes may also be used together,or with other enzymes, to thereby degrade sample components.

For example, the proteolytic enzyme trypsin is a serine protease thatcleaves peptide bonds between lysine or arginine and an unspecific aminoacid to thereby produce peptides that comprise an amine terminus(N-terminus) and lysine or arginine carboxyl terminal amino acid(C-terminus). In this way the peptides from the cleavage of the proteinare predictable and their presence and/or quantity, in a sample from atrypsin digest, can be indicative of the presence and/or quantity of theprotein of their origin. Moreover, the free amine termini of a peptidecan be a good nucleophile that facilitates its labeling. Other exemplaryproteolytic enzymes include papain, pepsin, ArgC, LysC, V8 protease,AspN, pronase, chymotrypsin and carboxypeptidase (e.g. carboxypeptidaseA, B, C, etc).

For example, a theoretical protein G might produce three peptides (e.g.peptides B, C and D) when digested with a protease such as trypsin.Accordingly, a sample that has been digested with a proteolytic enzyme,such as trypsin, and that when analyzed is confirmed to contain peptidesB, C and D, can be said to have originally comprised the protein G. Thequantity of peptides B, C and D will also correlate with the quantity ofthe protein G in the sample that was digested. In this way, anydetermination of the identity and/or quantify of one or more of peptidesB, C and D in a sample (or a fraction thereof), can be used to identifyand/or quantify the protein G in the original sample (or a fractionthereof).

Because activity of the enzymes is predictable, the sequence of peptidesthat are produced from degradation of a protein of known sequence can bepredicted (See above the discussion under the heading: “AnalyteDetermination By Computer Assisted Database Analysis”). With thisinformation, “theoretical” peptide information can be generated. Adetermination of the ‘theoretical” peptide fragments in computerassisted analysis of daughter fragment ions (as described above) frommass spectrometry analysis of an actual sample can therefore be used todetermine one or more peptides or proteins in one or more unknownsamples (Id.).

In some embodiments, sample processing can include treatment ofprecursors to the analyte or analytes to be labeled. For example, if theanalyte or analytes to be labeled are peptides derived from a digestedprotein and the labeling reagent is, for this example, selected to reactwith amine groups (e.g. N-α-amine groups and N-ε-amine group of lysine)of the peptide or peptide analytes, the protein (the analyte precursormolecule) of the sample may be processed in a manner that facilitatesthe labeling reaction. In this example, the protein can be reduced witha reducing agent (e.g. tris[2-carboxyethyl]phosphine (TCEP)) and thethiol groups then blocked by reaction with a blocking reagent (e.g.methyl methanethiosulfonate (MMTS)). In this way the thiol groups of theprotein are blocked and therefore do not interfere with the labelingreaction between the amines of the analytes and labeling reagent.

Those of skill in the art will appreciate that treatment of certainother precursor molecules can be performed using readily availablereagents and protocols that can be adapted with the aid of routingexperimentation. The precise choices or reagents and conditions can beselected depending on the nature of the analyte to be labeled and thelabeling reagent.

In some embodiments, sample processing can include the immobilization ofthe analytes or analyte precursors to a solid support, whether labeledwith a labeling reagent or not. Immobilization can include covalentimmobilization as well as adsorption and other non-covalent means ofimmobilization (e.g. electrostatic immobilization). In some embodiments,immobilization can facilitate reducing sample complexity. In someembodiments, immobilization can facilitate analyte labeling. In someembodiments, immobilization can facilitate analyte precursor labeling.In some embodiments, immobilization can facilitate selective labeling ofa fraction of sample components comprising a certain property (e.g. theycomprise or lack cysteine moieties). In some embodiments, immobilizationcan facilitate purification. The immobilization can facilitate two ormore of the foregoing.

Separation Including Separation of the Sample Mixture:

In some embodiments, the processing of a sample or sample mixture oflabeled analytes can involve separation. One or more separations can beperformed on the labeled and/or unlabeled analytes, labeled and/orunlabeled analyte precursors, or fractions thereof. One or moreseparations can be performed on one or more fractions obtained from asolid phase capture and/or other products of a separations process.Separations can be preformed on two or more of the foregoing sampletypes and different types of separations can be performed prior to massanalyses.

For example, a sample mixture comprising differentially labeled analytesfrom different samples can be prepared. By differentially labeled wemean that each of the labels comprises a unique property that can beidentified. The labels described herein can comprise two independentlydetectable properties. Specifically, they can comprise a non-encodeddetectable label as well as a unique reporter moiety that produces aunique “signature ion” in MS/MS (or MS^(n′) analysis). For example, inorder to analyze the sample mixture, components of the sample mixturecan be separated and mass analysis performed on only a fraction of thesample mixture. In this way, the complexity of the analysis can besubstantially reduced since separated analytes can be individuallyanalyzed for mass thereby increasing the sensitivity of the analysisprocess. Of course, the analysis can be repeated one or more time on oneor more additional fractions of the sample mixture to thereby allow forthe analysis of all fractions of the sample mixture and thus a morecomplete analysis of the components of the sample mixture.

Separation conditions under which identical analytes that aredifferentially labeled co-elute at a concentration, or in a quantity,that is in proportion to their abundance in the sample mixture can beused to determine the amount of each labeled analyte in each of thesamples that comprise the sample mixture provided that the amount ofeach sample added to the sample mixture is known. Accordingly, in someembodiments, separation of the sample mixture can simplify the analysiswhilst maintaining the correlation between signals determined in themass analysis (e.g. MS/MS analysis) with the amount of the differentlylabeled analytes in the sample mixture.

In some embodiments, a separation can be performed by chromatography.For example, liquid chromatography/mass spectrometry (LC/MS) can be usedto effect sample separation and mass analysis. Moreover, anychromatographic separation process suitable to separate the analytes ofinterest can be used. For example, the chromatographic separation can benormal phase chromatography, reversed-phase chromatography, ion-exchangechromatography (i.e. anion exchange chromatography or cation exchangechromatography), size exclusion chromatography or affinitychromatography.

When analyzing sample mixtures comprising analytes labeled with theisomeric and/or isobaric labeling reagents described herein,electrophoretic separation can be particularly useful. Non-limitingexamples of electrophoretic separations techniques that can be usedinclude, but are not limited to, 1D electrophoretic separation, 2Delectrophoretic separation and/or capillary electrophoretic separation(See: Westermeier, R., Electrophoresis in Practice: A Guide to Methodsand Applications of DNA and Protein Separations, Wiley-VCH Verlan GmbH,Weinheim, Germany, 2005 and M. Khaledi, M., High-Performance CapillaryElectrophoresis: Theory, Techniques, and Applications, John Wiley andSons, Inc. New York, 1998). During or after the separation, thenon-encoded detectable labels can be used to locate mixtures of labeledanalytes that co-elute, wherein the mixture can comprise an analyte ofinterest differentially labeled with different isomeric and/or isobariclabels wherein the each different label indicates from which sample theanalyte originated. Moreover, because the non-encoded detectable can beindependently detectable, the relative amounts of the labeled analytescan, in some embodiments, be determined and this information used tojudiciously select certain of the co-migrating labeled analytes (basedupon certain criteria) for further analysis by mass spectrometry,thereby avoiding the need to analyze all possible labeled analytespresent in the electrophoretic separation.

For example, the electrophoretic separation process can produce, forcertain analytes, a mixture comprising an amount of each isobaricallylabeled analyte that is in proportion to the amount of that labeledanalyte in the sample mixture. Furthermore, from the knowledge of howthe sample mixture was prepared (portions of samples and other optionalcomponents (e.g. calibration standards added to prepare the samplemixture)), it is possible to relate the amount of labeled analyte in thesample mixture back to the amount of that labeled analyte in the samplefrom which it originated. Based upon the relative amounts observed, itis also possible to select only those labeled analytes that meet acertain criteria of interest (e.g. are different in relative intensityby greater than 10%) for further analysis. For example, certain mixturesof isolated co-migrating analytes can also be subjected to analysis in amass spectrometer including analysis that provides quantificationinformation.

Relative and Absolute Quantification Of Analytes:

In some embodiments, the relative quantification of differentiallylabeled identical analytes of a sample mixture is possible. For example,relative quantification of differentially labeled identical analytes ispossible by comparison of the relative amounts (e.g. area and/or heightof the peak reported) of reporter ion (i.e. signature ion) that aredetermined in the mass analysis (e.g. in the second mass analysis for aselected, labeled analyte observed in a first mass analysis). Stateddifferently, where each reporter ion can be correlated with informationfor a particular sample used to produce a sample mixture, the relativeamount of that reporter ion, with respect to other reporter ionsobserved in the mass analysis, is the relative amount of that analyte inthe sample mixture. Where components combined to form the sample mixtureare known (e.g. the amount of each sample combined to form a samplemixture), the relative amount of the analyte in each sample used toprepare the sample mixture can be back calculated based upon therelative amounts of reporter ion observed for the labeled analyte ofselected mass to charge. This process can be repeated for all of thedifferent labeled analytes observed in the first mass analysis. In thisway, the relative amount (often expressed in concentration and/orquantity) of each reactive analyte, in each of the different samplesused to produce the sample mixture, can be determined.

In other embodiments, absolute quantification of analytes can bedetermined. For these embodiments, a known amount of one or moredifferentially labeled analytes (the calibration standard or calibrationstandards) can be added to the sample mixture or the intensity of thereporter ion can be correlated with a calibration curve.

A calibration standard can be an expected analyte that is labeled withan isomeric and/or isobaric label of the set of labels used to label theanalytes of the sample mixture provided that the reporter moiety for thecalibration standard is unique as compared with any of the samples usedto form the sample mixture. Once the relative amount of reporter ion forthe calibration standard, or standards, is determined with relation tothe relative amounts of the reporter ion or ions for the differentiallylabeled analytes of the sample mixture, it is possible to calculate theabsolute amount (often expressed in concentration and/or quantity) ofall of the differentially labeled analytes in the sample mixture withreference to the amount of calibration standard or standards that was(were) added to the sample mixture. In this way, the absolute amount ofeach differentially labeled analyte (for which there is a calibrationstandard in the sample from which the analyte originated) can also bedetermined based upon the knowledge of how the sample mixture wasprepared.

Alternatively, a calibration curve can be prepared by analysis ofrepresentative samples of labeled analytes, each sample comprising adifferent known amount of the labeled analyte. The intensities of thepeaks of the reporter ion for the analyzed labeled analyte can beplotted with respect to the known amount of each labeled analyte tothereby generate the standard curve. Once prepared the intensity of areporter ion in an unknown sample can be compared with the standardcurve to thereby determine the amount of the analyte in a test sample.

Notwithstanding the foregoing, corrections to the intensity of thereporters ion (signature ions) can be made, as appropriate, for anynaturally occurring, or artificially created, isotopic abundance withinthe reporter moieties. There are numerous ways to correct for isotopicabundance of impurities in the signature ions of reporter moieties. Anexample of such a correction can be found in published copending andco-owned United States Provisional Patent Application No. US2005-0114042 A1, entitled: “Method and Apparatus For De-Convoluting AConvoluted Spectrum”, filed on Aug. 12, 2004. Basically, the intensityof up-mass and down mass peaks associated with the isotopic cluster of asingle labeling reagent can be determined by deconvolution of theconvoluted spectrum of the overlapping isotopic clusters of the labelingreagents using mathematical formulas and calculations. Regardless of howthe values are determined, the more care taken to accurately quantifythe intensity of each reporter ion (i.e. signature ion), the moreaccurate will be the relative and absolute quantification of theanalytes in the original samples.

Proteomic Analysis:

Embodiments of this invention can be used for complex analysis becausesamples can be multiplexed, analyzed and reanalyzed in a rapid andrepetitive manner using mass analysis techniques. For example, samplemixtures can be analyzed for the amount of one or more analytes in oneor more samples. The amount (often expressed in concentration and/orquantity) of the analyte or analytes can be determined for the samplesfrom which the sample mixture was comprised. Because the sampleprocessing and mass analyses can be performed rapidly, these methods canbe repeated numerous times so that the amount of many differentiallylabeled analytes of the sample mixture can be determined with regard totheir relative and/or absolute amounts in the sample from which theanalyte originated.

One application where such a rapid multiplex analysis is useful is inthe area of proteomic analysis. Proteomics can be viewed as anexperimental approach to describe the information encoded in genomicsequences in terms of structure, function and regulation of biologicalprocesses. This may be achieved by systematic analysis of the totalprotein component expressed by a cell or tissue. Mass spectrometry, usedin combination with the method, mixture, kit and/or compositionembodiments of this invention is one possible tool for such globalprotein analysis. Accordingly, experimental analysis for which theselabeling reagents can be used includes, but is not limited to, timecourse experiments, biomarker analysis, multiplex proteomic analysis,mudpit experiments, affinity pull-downs, determination ofpost-translational modifications (PTMs) and multiple controlexperiments.

II Compositions

In some embodiments, this invention pertains to compounds represented byformula I;

including a salt form and/or hydrate form thereof, wherein m and n areeach 0 or 1 provided that m+n=1 and wherein the compound can beisotopically encoded with one or more heavy atom isotopes in thereporter moiety and/or linker moiety. As described in more detail above,the reactive group “RG” can comprising a nucleophile or an electrophilewherein said reactive group is capable of reacting with one or morereactive analytes of a sample to thereby form one or more labeledanalytes. As described in more detail above, the reporter moiety “RP”can comprise a fixed charge or be ionizable in a mass spectrometer,wherein said reporter moiety is capable of producing a signature ion ina mass spectrometer upon fragmentation of bond X. As described in moredetail above, “DL” can be a non-encoded detectable label. As describedin more detail above, the linker moiety “LK” can be linear or branched,wherein LK links the reactive group to both the reporter moiety and thenon-encoded detectable label. As described in more detail above,covalent bond X links the reporter moiety to the linker moiety or to thenon-encoded detectable label wherein bond X is fragmentable byapplication of dissociative energy in a mass spectrometer. As describedin more detail above, Y can be a covalent bond or linking group thatlinks the reactive group to the linker moiety, wherein Y is optionallyfragmentable by application of dissociative energy in a massspectrometer. As described in more detail above, W can be a covalentbond or linking group that links the non-encoded detectable label to thelinker moiety, wherein W is optionally cleavable by application oflight, heat and/or chemical reagent(s) and/or fragmentable byapplication of dissociative energy in a mass spectrometer. In someembodiments, the compound is optionally linked to a solid support via acleavable linker. In some embodiments, the compound is linked to thesolid support through the reporter moiety. Certain fragmentationproperties of labeling reagents of formula I are illustrated in FIG. 2a.

For example, the composition can be a compound represented by formula IIor III;

wherein RG is the reactive group and wherein the compound can beisotopically encoded (c.f. FIG. 8 a). Compositions of formulas I, II orIII can be used to label analytes for their analysis in methods that canutilize electrophoretic separations techniques in combination with massspectrometry (e.g. tandem mass spectrometry) as discussed in more detailherein. The elements and certain fragmentation properties of labelingreagents of formula II and III are illustrated in FIG. 2 b.

The compositions can comprise an isotopically enriched (i.e. encoded)reporter moiety and/or an isotopically enriched linker moiety. Forexample, the reporter moiety and/or linker moiety can each beisotopically enriched to comprise one or more heavy atom isotopes. Insome embodiments, the reporter moiety and/or linker moiety can each beisotopically enriched to comprise two or more heavy atom isotopes. Insome embodiments, the reporter moiety and/or linker moiety can each beisotopically enriched to comprise three or more heavy atom isotopes. Insome embodiments, the reporter moiety and/or linker moiety can each beisotopically enriched to comprise four or more heavy atom isotopes.

In some embodiments, the reporter moiety can be cleavably linked to asupport. By “cleavably linked” we mean that the reporter moiety, andanything covalently linked thereto, can be released from the support bytreatment with an appropriate agent, such as light, heat and/or chemicalreagent(s), based upon the nature of the cleavable linker chosen.Various supports comprising cleavable linkers are well known in the art.For example, various supports comprising a trityl moiety are soldcommercially or can otherwise be prepared (e.g. Trityl chloride support(Trityl-Cl) or 2-Chlorotrityl chloride support). With reference to FIG.6, an embodiment of a support-bound labeling reagent is illustratedwherein the reporter moiety of the labeling reagent comprises abis-N-alkylated piperazine ring wherein one of the substituents of theN-alkylated piperazine ring is an alkyl group terminating with groupfunctional Q, wherein Q is a functional group that can be released upontreatment of the support with acid to thereby produce the labeledanalyte.

Thus, in some embodiments, this invention pertains to labeling reagentcompositions represented by formula I^(ss);

including a salt form and/or hydrate form thereof, wherein m and n areeach 0 or 1 provided that m+n=1 and wherein the compound can beisotopically encoded with one or more heavy atom isotopes in thereporter moiety and/or linker moiety. As described in more detail above,the reactive group “RG” can comprising a nucleophile or an electrophilewherein said reactive group is capable of reacting with one or morereactive analytes of a sample to thereby form one or more labeledanalytes. As described in more detail above, the reporter moiety “RP”can comprises a fixed charge or be ionizable in a mass spectrometer,wherein said reporter moiety is capable of producing a signature ion ina mass spectrometer upon fragmentation of bond X. As described in moredetail above, “DL” can be a non-encoded detectable label. As describedin more detail above, the linker moiety “LK” can be linear or branched,wherein LK links (directly or indirectly) the reactive group to both thereporter moiety and the non-encoded detectable label (c.f. FIGS. 1 a and1 b). As described in more detail above, covalent bond X links thereporter moiety to the linker moiety or to the non-encoded detectablelabel wherein bond X is fragmentable by application of dissociativeenergy in a mass spectrometer. As described in more detail above, Y canbe a covalent bond or linking group that links the reactive group to thelinker moiety, wherein Y is optionally fragmentable by application ofdissociative energy in a mass spectrometer. As described in more detailabove, W can be a covalent bond or linking group that links thenon-encoded detectable label to the linker moiety, wherein W isoptionally cleavable by application of light, heat and/or chemicalreagent(s) and/or fragmentable by application of dissociative energy ina mass spectrometer. As described in more detail above, SS is a solidsupport to which the reporter moiety of the composition is covalentlylinked through a cleavable linker. Certain fragmentation properties oflabeling reagent of formula I^(ss) are illustrated in FIG. 2 a.

For example, the composition can be a compound represented by formulaII^(ss) or III^(ss);

wherein RG is the reactive group, SS is the solid support and CL is thecleavable linker and wherein the compound can be isotopically encodedwith one or more heavy atom isotopes in the reporter moiety and/orlinker moiety. The elements and certain fragmentation properties oflabeling reagents of formulas II^(ss) and III^(ss) are illustrated inFIG. 2 c.

In some embodiments, the group RG of a compounds of formulas I, II, III,I^(ss), II^(ss) or III^(ss), described above, can be a carboxylic acid,a sulfonic acid, a carboxylic acid halide, a sulfonyl halide, an activeester, a mixed anhydride, an isocyanate or an isothiocyanate group. Insome embodiments, the group RG can be a malemide group, an alkyl halidegroup, an α-halo-acyl group, an α-halo thione group or an α-halo iminegroup. In some embodiments, the group RG can be a trityl-halide or asilyl-halide group. In some embodiments, the group RG can be an aminegroup, a hydroxyl group or a thiol group.

As stated, the compositions described above can exist in a salt formand/or hydrate form. Whether or not the composition exists as a saltform will typically depend upon the nature and number of substituents aswell as the conditions under which it exists and/or was isolated. It iswell known that basic groups such as amines can be protonated bytreatment with acid to thereby form salts of the amine. For example,piperazine containing labeling reagents can be obtained as a mono-TFAsalt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt (See forExample, US Patent Application Publication No. US 2005-0148771 A1). Oneof ordinary skill in the art will surely appreciate how to manipulatethe charge state and nature of any counterion the salt form of thecompositions disclosed herein using no more than routine experimentationand the disclosure provided herein.

Whether or not a composition exists as a hydrate will also depend on theconditions under which it exists or was isolated. Hydrates merelycomprise one or more complexed water molecules. This inventioncontemplates any possible hydrate form.

In some embodiments, this invention also pertains to analytes that arelabeled with the labeling reagent compositions disclosed herein. Thus,in some embodiments, this invention pertains to labeled analytecompositions represented by formula I^(A);

including a salt form and/or hydrate form thereof, wherein m and n areeach 0 or 1 provided that m+n=1 and wherein RP, X, W and DL are asdescribed for the composition of formula I, above and wherein thecompound can be isotopically encoded with one or more heavy atomisotopes in the reporter moiety and/or linker moiety. As described inmore detail above, the linker moiety “LK” can be linear or branched,wherein LK links (directly or indirectly) the analyte to both thereporter moiety and the non-encoded detectable label. As described inmore detail above, Y can be a covalent bond or linking group that linksthe analyte to the linker moiety, wherein Y is optionally fragmentableby application of dissociative energy in a mass spectrometer.

In some embodiments, this invention also pertains to support-boundlabeled analyte compositions represented by formula I^(ssA);

including a salt form and/or hydrate form thereof, wherein m and n areeach 0 or 1 provided that m+n=1 and wherein SS, RP, X, W and DL are asdescribed for the composition of formula I^(ss), above and wherein thecompound can be isotopically encoded with one or more heavy atomisotopes in the reporter moiety and/or linker moiety. As described inmore detail above, the linker moiety “LK” can be linear or branched,wherein LK links (directly or indirectly) the analyte to both thereporter moiety and the non-encoded detectable label. As described inmore detail above, Y can be a covalent bond or linking group that linksthe analyte to the linker moiety, wherein Y is optionally fragmentableby application of dissociative energy in a mass spectrometer. Certainfragmentation properties of support-bound labeled analyte I^(ssA) isillustrated in FIG. 3 a.

For example, the support-bound labeled analyte can be represented byformula II^(ssA) or III^(ssA).

wherein SS is the solid support and CL is the cleavable linker. Theelements and certain fragmentation properties of labeled analytesII^(ssA) and III^(ssA) are illustrated in FIG. 3 c.

Analytes have been previously described herein. In some embodiments, thelabeled analyte can be a labeled calibration standard. As describedherein, calibration standards can be added to mixtures in knownquantities to facilitate absolute quantitative analysis of an analyte ofinterest. Accordingly, in some embodiments, this invention pertains toan analyte, such as a peptide, protein, nucleic acid, carbohydrate,steroid, lipid, amino acid, vitamin or prostaglandin of interest, whichhas been labeled with a labeling reagent composition as described abovefor the purpose of being a calibration standard. Thus, the labeledcalibration standard can be any analyte labeled with a labeling reagentas described herein. Typically, the labeling reagent is selected from aset of isomeric and/or isobaric labeling reagents so that it comprises aunique reporter moiety as compared with the labeling reagents used tolabel one or more test samples of interest for which quantification ofsaid analyte in said test samples is of interest.

III. Methods for Labeling and Analysis

According to some embodiments of this invention, analytes can be labeledwith labeling reagents described above and then determined. The labeledanalyte, the analyte itself, one or more fragments of the analyte and/orfragments of the label, can be determined by mass analysis. In someembodiments, methods of this invention can be used for the analysis ofdifferent analytes in the same sample as well as for the multiplexanalysis of the same and/or different analytes in two or more differentsamples. The two or more samples can be mixed to form a sample mixture.In multiplex analysis, labeling reagents can be used to determine fromwhich sample of a sample mixture an analyte originated. The absoluteand/or relative (with respect to the same analyte in different samples)amount (often expressed in concentration or quantity) of the analyte, ineach of two or more of the samples combined to form the sample mixture,can be determined. Moreover, mass analysis of fragments of the analyte(e.g. daughter fragment ions) can be used to identify the analyte and/orthe precursor to the analyte; such as where the precursor molecule tothe analyte was degraded.

The samples used in the analysis may be any sample comprising an analyteor analytes that can be labeled with isotopically encoded versions ofthe labeling reagents. For example, the sample can be a crude orprocessed cell lysate, a body fluid, a tissue extract or a cell extract.In some embodiments, the sample can be processed before labeling tothereby prepare the analytes, or other components of the sample, for thelabeling reaction. The sample can be a fraction from a separationsprocess. The analyte in the sample can be any analyte that can belabeled with the labeling reagent. For example, the analyte can be apeptide, a protein, a nucleic acid, a carbohydrate, a lipid, a steroid,an amino acid, a vitamin, a prostaglandin or other small molecule with amolecular weight of less than 1500 daltons (Da). Other possible analytetypes have been disclosed herein.

Consequently, in some embodiments, this invention also pertains to amethod comprising reacting two or more samples, each sample comprisingone or more reactive analytes, with a different labeling reagent of aset of labeling reagents to thereby produce two or more differentiallylabeled samples each comprising one or more labeled analytes. Thelabeling reagents can be selected from a set of isotopically encodedisomeric and/or isobaric labeling reagents wherein the differentlabeling reagents each comprise a reporter moiety of unique mass. Thereporter moiety can be any reporter moiety having the propertiesdisclosed herein. For example, the reporter moiety can comprise asubstituted or unsubstituted piperidine, piperazine or morpholine group.Examples of other reporter moieties are also described above.

In some embodiments, this invention pertains to reacting two or moresamples, each sample comprising one or more reactive analytes, with adifferent labeling reagent of a set of isotopically enriched isomericand/or isobaric labeling reagents to thereby form two or moredifferentially labeled samples each comprising one or more labeledanalytes; wherein the different labeling reagents of the set arerepresented by formula I;

including a salt form and/or hydrate form thereof, wherein m and n areeach 0 or 1 provided that m+n=1. According to the method, RG (asdescribed in more detail above) is a reactive group comprising anucleophile or an electrophile and said reactive group reacts with theone or more reactive analytes of the sample to thereby form the one ormore labeled analytes. The reporter moiety “RP” (as described in moredetail above) comprises a fixed charge or is ionizable in a massspectrometer, wherein said reporter moiety is capable of producing asignature ion in a mass spectrometer upon fragmentation of bond X andwherein the gross mass of each reporter moiety, and its correspondingsignature ion, is different for each labeling reagent of the set. Thenon-encoded detectable label “DL” (as described in more detail above) isa different independently detectable non-encoded detectable label foreach reagent of the set, wherein each non-encoded detectable label hasthe same gross mass and same net charge as the other non-encodeddetectable labels of the labeling reagents of the set. The linker moiety“LK” (as described in more detail above) can be linear or branched,wherein LK links (directly or indirectly) the reactive group to both thereporter moiety and the non-encoded detectable label and wherein themass of the linker moiety of each different reagent of the setcompensates for the difference in gross mass between the reportermoieties for the different labeling reagents of the set such that theaggregate gross mass of the combination for the reporter moiety and thelinker moiety is the same for each labeling reagent of the set. Thecovalent bond X (as described in more detail above) links the reportermoiety to the linker moiety or to the non-encoded detectable label,wherein X is fragmentable by application of dissociative energy in amass spectrometer. Y (as described in more detail above) is a covalentbond or linking group that links the reactive group to the linkermoiety, wherein Y is optionally fragmentable by application ofdissociative energy in a mass spectrometer, provided that if Y is alinking group its gross mass is the same for every labeling reagent ofthe set. W (as described in more detail above) is a covalent bond orlinking group that links the non-encoded detectable label to the linkermoiety, wherein W is optionally cleavable by application of light, heatand/or chemical reagent(s) and/or fragmentable by application ofdissociative energy in a mass spectrometer, provided that if W is alinking group its gross mass is the same for every labeling reagent ofthe set.

In some embodiments, each differentially labeled sample is labeled witha labeling reagent of formula II or formula III;

wherein RG is the reactive group. In some embodiments, compounds II andIII can be isotopically encoded as shown in FIG. 8 a.

The labeling process can produce two or more differentially labeledsamples each comprising one or more labeled analytes. Once the analytesof each sample are labeled with the labeling reagent that is unique tothat sample, two or more of the differentially labeled samples, or aportion thereof, can be mixed to produce a sample mixture. The samplemixture can optionally comprise one or more calibration standards asdescribed above.

The volume and/or quantity of each sample of labeled analytes combinedto produce the sample mixture can be recorded. The volume and/orquantity of each sample, relative to the total sample volume and/orquantity of the sample mixture, can be used to determine a ratio thatcan be used for determining the amount (often expressed in concentrationand/or quantity) of an identified analyte in each sample from theanalysis of the sample mixture. The sample mixture can thereforecomprise a complex mixture wherein relative amounts of the same and/ordifferent analytes can be identified and/or quantified, either byrelative quantification of the amounts of analyte in each of the two ormore samples or absolutely where a calibration standard is also added tothe sample mixture or where a calibration curve for the signature ionsis available.

In some embodiments, the method can further comprise separatingelectrophoretically the sample mixture, or a portion thereof. Forexample, the electrophoretic separation can be a 1D electrophoreticseparation, a 2D electrophoretic separation and/or a capillaryelectrophoretic separation.

The two or more non-encoded detectable labels can be independentlydetectable. Because the labels can be independently detectable, it ispossible to locate, detect and/or quantify labeled analytes during theelectrophoretic separation. For example, the independently detectablenon-encoded detectable labels can be used to locate labeled analytes ina 1-D or 2-D gel during and/or after separation. In some embodiments,the labels can be used to locate the labeled analytes in the gel afterseparation so that they can be excised from the gel and furtheranalyzed, such as by mass spectrometry as discussed below.

For example the method can further comprise collecting one or moresub-samples of co-migrating differentially labeled analytes from theelectrophoretic separation and optionally treating the one or moresub-samples under appropriate conditions to thereby cleave thenon-encoded detectable label from the differentially labeled analytes ofthe sub-sample or sub-samples where m is 0, n is 1 and W is a covalentbond or linking group that is cleavable by application of light, heat orchemical reagent(s). Once the non-encoded detectable label has beencleaved from the analytes, they can be further analyzed, such as by massspectrometry, wherein analysis of reporter moieties can be used todetermine and/or confirm the relative and/or absolute quantity of theanalyte associated with each sample used to form the sample mixture asdiscussed below.

In some embodiments, the non-encoded detectable labels can be used incapillary electrophoresis to detect and/or quantify labeled analytes asthey exit the capillary column. Quantification can be determined basedupon an analysis of the intensity of signal generated from thenon-encoded detectable labels in a suitable detector. Preferentially(but not essentially), the non-encoded detectable labels of a set areselected to have similar migration properties (i.e. they co-migrate)during the electrophetic separation. In some embodiments, theco-migrating differentially labeled analytes can optionally be analyzed,based upon the independently detectable properties of the non-encodeddetectable labels, for a condition of interest and judiciously selectedfor further analysis based upon satisfaction of the condition else bediscarded based upon a failure to satisfy the condition. For example, ifthe co-migrating differentially labeled analytes satisfy the conditionof interest (e.g. the relative amounts of the two independentlydetectable labels differs by more than 10%), the co-migratingdifferentially labeled analytes can be subsequently analyzed andquantified by mass spectrometry but if they don't, they are discarded.In this way, it is possible to achieve more efficient and economical useof the mass spectrometry equipment.

In some embodiments, the method can further comprise performing a firstmass spectrometric analysis on one of the sub-samples, or a fractionthereof, using a first mass analyzer. Ions of a particular mass tocharge ratio from the first mass analysis can then be selected. Theselected ions can be subjected to dissociative energy levels (e.g.collision-induced dissociation (CID)) to thereby induce fragmentation.For example, fragmentation of bond X can cause release the ionizedreporter moiety (i.e. the reporter ion or signature ion) from thelabeled analyte. Fragmentation of the selected ions by the dissociativeenergy can also produce daughter fragment ions of the analyte that canbe subsequently analyzed to determine the analyte and/or one or moreprecursors to the analyte. Thus, the ions (remaining selected ions,daughter fragment ions and ionized reporter moieties (i.e. signatureions)), or a fraction thereof, can then be directed to a second massanalyzer for analysis.

In the second mass analyzer, a second mass analysis can be performed onthe selected ions, the signature ions and the daughter fragment ions ora fraction thereof. The second mass analysis can determine the grossmass (or m/z) and relative amount of each unique reporter ion that ispresent at the selected mass to charge ratio as well as the mass (grossand/or absolute) of some or all of the daughter fragment ions of atleast one labeled analyte of the sample mixture. For each analytepresent at the selected mass to charge ratio, the daughter fragment ionscan be used to identify the analyte and/or analytes present at theselected mass to charge ratio. For example, this analysis can be done aspreviously described in the section entitled: “Analyte Determination ByComputer Assisted Database Analysis”. Thus, in some embodiments, themethod further comprises determining the gross mass and relative amountof each signature ion in the second mass analysis and the gross and/orabsolute mass of some or all of the daughter fragment ions in the secondmass analysis. In some embodiments, the method further comprisesdetermining the labeled analyte (and/or a precursor thereto) associatedwith the selected mass to charge ratio by analysis of the daughterfragment ions.

In some embodiments, one or more steps of the process can be repeatedone or more times. For example, in some embodiments, ions of a selectedmass to charge ratio from the first mass spectrometric analysis,different from any previously selected mass to charge ratio, can betreated to dissociative energy to thereby form ionized reporter moieties(i.e. signature ions) and daughter fragment ions of at least some of theselected ions, as previously described. A second mass analysis of theselected ions, the reporter ions and/or the daughter fragment ions, or afraction thereof, can be performed. The gross mass and relative amountof each unique signature ion in the second mass analysis and the mass(gross or absolute) of the daughter fragment ions can also bedetermined. Optionally, the labeled analyte (or precursor molecule)associated with the selected mass to charge ratio can be determined byanalysis of the daughter fragment ions. In this way, the information canbe made available for identifying and/or quantifying one or moreadditional analytes from the first mass analysis.

In some embodiments, it may be useful to repeat the process (or certainsteps of the method) one or more times on a different collectedsub-sample where the sample mixture has been fractionated (e.g. byelectrophoretic separation). By repeating the processes on one or moreadditional fractions of the sample, it is possible to more fully analyzethe sample mixture. Consequently, the method can comprise selecting adifferent sub-sample and performing a first mass analysis on thesub-sample and then repeating one or more of the subsequent stepspreviously described.

In some embodiments, it may be useful to repeat the process one or moretimes by collecting one or more different sub-samples of co-migratingdifferentially labeled analytes from the electrophoretic separationwhere the sample mixture has been fractionated. By repeating theprocesses to thereby collect one or more additional sub-samples, it ispossible to more fully analyze the sample mixture. Consequently, themethod can comprise collecting one or more different sub-samples ofco-migrating differentially labeled analytes from the electrophoreticseparation and then repeating one or more of the subsequent previouslydescribed steps.

It is contemplated that in some embodiments, the whole process will berepeated one or more times and within each of these repeats, certainsteps can also be repeated one or more times such as described above. Inthis way, the contents of the sample mixture can be interrogated anddetermined to the fullest possible extent. In some embodiments, theentire process can also be repeated on a new set of two or more samples.

As previously discussed, in some embodiments, the labeling reagents ofthe set of isomeric and/or isobaric labeling reagents can besupport-bound. Accordingly, except for accommodating recapture of thelabeled analytes from the support, the above described methods can bepracticed with the support-bound reagents. This, in some embodiments,this invention pertains to practicing any of the above disclosedmethods, wherein each different labeling reagent of the set issupport-bound and is linked to the support through a cleavable linkersuch that each different sample is reacted with a support carrying adifferent labeling reagent of the set and wherein the method furthercomprises, after performing the step of labeling the sample but beforeperforming the step of mixing the labeled samples to prepare the samplemixture: i) optionally washing each support to remove components of eachsample that do not react with the reactive group of the support-boundlabeling reagent; ii) cleaving the cleavable linker to thereby releasethe labeled analytes from each support, each differentially labeledsample comprising one or more labeled analytes wherein the labeledanalytes associated with a particular sample are identifiable and/orquantifiable by the reporter moiety of unique mass linked thereto; andiii) optionally collecting the labeled analytes of each sample prior tomixing them according to step (b).

For example, the method can be practiced with a set of isotopicallylabeling reagents wherein each different labeling reagent of the set isrepresented by formula I^(ss);

including a salt form and/or hydrate form thereof; wherein m and n areeach 0 or 1 provided that m+n=1. According to the method, RG (asdescribed in more detail above) is a reactive group comprising anucleophile or an electrophile and said reactive group reacts with theone or more reactive analytes of the sample to thereby form the one ormore labeled analytes. The reporter moiety “RP” (as described in moredetail above) comprises a fixed charge or is ionizable in a massspectrometer, wherein said reporter moiety is capable of producing asignature ion in a mass spectrometer upon fragmentation of bond X andwherein the gross mass of each reporter moiety, and its correspondingsignature ion, is different for each labeling reagent of the set. Thenon-encoded detectable label “DL” (as described in more detail above) isa different independently detectable non-encoded detectable label foreach reagent of the set, wherein each non-encoded detectable label hasthe same gross mass and same net charge as the other non-encodeddetectable labels of the labeling reagents of the set. The linker moiety“LK” (as described in more detail above) can be linear or branched,wherein LK links (directly or indirectly) the reactive group to both thereporter moiety and the non-encoded detectable label and wherein themass of the linker moiety of each different reagent of the setcompensates for the difference in gross mass between the reportermoieties for the different labeling reagents of the set such that theaggregate gross mass of the combination of the reporter moiety and thelinker moiety is the same for each labeling reagent of the set. Thecovalent bond X (as described in more detail above) links the reportermoiety to the linker moiety or to the non-encoded detectable label,wherein X is fragmentable by application of dissociative energy in amass spectrometer. Y (as described in more detail above) is a covalentbond or linking group that links the reactive group to the linkermoiety, wherein Y is optionally fragmentable by application ofdissociative energy in a mass spectrometer, provided that if Y is alinking group its gross mass is the same for every labeling reagent ofthe set. W (as described in more detail above) is a covalent bond orlinking group that links the non-encoded detectable label to the linkermoiety, wherein W is optionally cleavable by application of light, heatand/or chemical reagent(s) and/or fragmentable by application ofdissociative energy in a mass spectrometer, provided that if W is alinking group its gross mass is the same for every labeling reagent ofthe set. SS (as described in more detail above) is a solid support towhich the reporter moiety of the labeling reagent is covalently linkedthrough a cleavable linker.

In some embodiments, one of the labeling reagents can be represented byformula II^(ss) or III^(ss);

wherein RG is the reactive group, SS is the solid support and CL is thecleavable linker. In some embodiments, compounds II^(ss) and III^(ss)can be isotopically encoded as illustrated in FIG. 8 b.

Those of ordinary skill in the art of mass spectrometry will appreciatethat the first and second mass analysis described in the above disclosedmethods can be performed in a tandem mass spectrometer. Instrumentssuitable for performing tandem mass analysis have been previouslydescribed herein. Although tandem mass spectrometers are preferred,single-stage mass spectrometers may also be used. For example, analytefragmentation may be induced by cone-voltage fragmentation, followed bymass analysis of the resulting fragments using a single-stage quadrupoleor time-of-flight mass spectrometer. In other examples, analytes may besubjected to dissociative energy levels using a laser source and theresulting fragments recorded following post-source decay intime-of-flight or tandem time-of-flight (TOF-TOF) mass spectrometers.

In some embodiments, methods of the invention can further comprisedigesting each sample and/or the sample mixture with at least one enzymeto partially, or fully, degrade components of the sample and/or thesample mixture prior to performing the labeling of the analytes of thesample (Also see the above section entitled: “Sample Processing”). Forexample, the enzyme can be a protease (to degrade proteins and/orpeptides) or a nuclease (to degrade nucleic acids). Two or more enzymesmay also be used together to thereby further degrade sample components.For example, the enzyme can be a proteolytic enzyme such as trypsin,papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin ora carboxypeptidase (e.g. A, B, C, etc).

In some embodiments, methods can further comprise separating the samplemixture by application of an additional separations process other thanelectrophoresis prior to performing the first mass analysis (Also seethe above section entitled: “Separation Including Separation Of TheSample Mixture”). For example, liquid chromatography/mass spectrometry(LC/MS) can be used to effect such a sample separation prior to the massanalysis.

In some embodiments, the methods can be practiced with digestion andadditional separation steps. While these steps are optional, they can beperformed together, for example, when proteomic analysis is being doneto thereby determine the up and down regulation of proteins in cells. Insome embodiments, the steps of the methods, with or without thedigestion and/or separation steps, can be repeated one or more times tothereby identify and/or quantify one or more other analytes in a sampleor one or more analytes in each of the two or more samples (includingsamples labeled with support-bound labeling reagents). Depending ofwhether or not a calibration standard is present in the sample mixtureor whether or not a calibration curve for the signature ion isavailable, the quantification of a particular analyte can be relative tothe other labeled analytes, or it can be absolute.

As described previously, it is possible to determine the analyteassociated with the selected ions by analysis of the mass (gross orabsolute) of the daughter fragment ions. One such method ofdetermination is described in the section entitled: “AnalyteDetermination By Computer Assisted Database Analysis”. Once the analytehas been determined, information regarding the gross mass and relativeamount of each unique reporter ion in the second mass analysis and themass of daughter fragment ions provides the basis to determine otherinformation about the sample mixture.

The relative amount of signature ion (reporter ion) can be determined bypeak intensity in the mass spectrum. In some embodiments, the amount ofeach unique signature ion can be determined by analysis of the peakheight or peak width (or peak area) of the reporter ion (signature ion)obtained using a mass spectrometer. Because each sample can be labeledwith a different labeling reagent and each labeling reagent can comprisea unique reporter moiety that produces a unique signature ion that canbe correlated with a particular differentially labeled sample used toformulate the sample mixture, determination of the different reporterions in the second mass analysis can be used to identify thedifferentially labeled sample from which the signature ions of theselected analyte originated. Where multiple signature ions are found(e.g. according to the multiplex methods of the invention), the relativeamount of each unique signature ion can be determined with respect tothe other signature ions. Because the relative amount of each uniquesignature ion determined in the second mass analysis can be correlatedwith the relative amount of an analyte in the sample mixture, therelative amount (often expressed as concentration and/or quantity) ofthe analyte in each of the differentially labeled samples combined toform the sample mixture can be determined. Moreover, it is possible torelate the quantification information for an analyte to components ofthe original differentially labeled samples where an analyte that isdetermined is a by-product from another compound of interest (e.g. theanalyte is a product of a degradation reaction such as where the analyteis a peptide formed by the digestion of a protein).

As discussed above, this analysis can be repeated one or more times onselected ions of a different mass to charge ratio to thereby obtain therelative amount of one or more other determined analytes in each samplecombined to form the sample mixture. Moreover, as appropriate, acorrection of peak intensity associated with each unique signature ioncan be performed for naturally occurring, or artificially created,isotopic abundance, as previously discussed in the section entitled:“Relative and Absolute Quantification of Analytes”.

For example, the analytes can be peptides in a sample or sample mixture.Analysis of the peptides in a sample, or sample mixture, can be used todetermine the amount (often expressed as a concentration and/orquantity) of identifiable proteins in the sample or sample mixturewherein proteins in one or more samples can be degraded prior to thefirst mass analysis and wherein the amount of protein in the sample isdetermined based upon the identity and relative amount of the one ormore peptides in each of the two or more differentially labeled samplesmixed to form the sample mixture. Moreover, the information fromdifferent samples can be compared for the purpose of makingdeterminations, such as for the comparison of the effect on the amountof the protein in cells that are incubated with differing concentrationsof a substance that may affect cell growth, development, differentiationand/or death. Other, non-limiting examples may include comparison of theexpressed protein components of diseased and healthy tissue or cellcultures. This may encompass comparison of expressed protein levels incells, tissues or biological fluids following infection with aninfective agent such as a bacteria or virus or other disease states suchas cancer. In other examples, changes in protein concentration over time(time-course) studies may be undertaken to examine the effect of drugtreatment on the expressed protein component of cells or tissues. Instill other examples, the information from different samples taken overtime may be used to detect and monitor the concentration of specificproteins in tissues, organs or biological fluids as a result of disease(e.g. cancer) or infection. Such experiments may include one or morecontrol samples. In some embodiments, the experiments can be used todetermine two or more of the characteristics of interest describedabove.

Where a calibration standard comprising a unique signature moiety linkedto an analyte, having the selected mass to charge ratio, has been addedto the sample mixture in a known amount (often expressed as aconcentration and/or quantity) for the determined analyte, the amount ofthe unique signature ion associated with the calibration standard can beused to determine the absolute amount (often expressed as aconcentration and/or quantity) of the analyte in each of the samplescombined to form the sample mixture. This is possible because the amountof analyte associated with the unique signature ion for the calibrationstandard in the sample mixture is known and the relative amounts of allunique signature ions can be determined for the labeled analyteassociated with the selected ions with reference to the intensity of thesignature ion of the calibration standard. Because the relative amountof each unique signature ion, determined for each of the uniquereporters moieties (including the reporter moiety for the calibrationstandard), is proportional to the amount of the analyte associated witheach differentially labeled sample combined to form the sample mixture,the absolute amount (often expressed as a concentration and/or quantity)of the analyte in each of the samples can be determined with referenceto the amount of each different signature ion of unique mass based upona ratio calculated with respect to the formulation used to produce thesample mixture. As appropriate, a correction of peak intensityassociated with each of the unique signature ions can be performed fornaturally occurring, or artificially created, isotopic abundance. Suchan analysis method can be particularly useful for proteomic analysis ofmultiplex samples of a complex nature, especially where a preliminaryseparation of the labeled analytes (e.g. liquid chromatography orelectrophoretic separation) precedes the first mass analysis.

For example, if a sample mixture comprises 100 fmol/mL of a calibrationstandard and the relative intensity of the unique signature ionassociated with the calibration standard was 1 while the relativeintensity of a first other unique signature ion associated with a firstsample was one-half and the relative intensity of a second other uniquesignature ion associated with a second sample was 2, the amount of theanalyte in the first differentially labeled sample mixed to form thesample mixture (assuming equal amounts of sample 1 and sample 2 aremixed to form the sample mixture) is 50 fmol/mL (0.5×100 fmol/mL) andthe amount of the analyte in the second differentially labeled samplemixed to form the sample mixture is 200 fmol/mL (2×100 fmol/mL).Moreover, if, for example, the analyte is a peptide associated with aparticular protein, it can be inferred that the amount of the protein insample 1 is 50 fmol/mL and the amount of the protein in sample 2 is 200fmol/mL. Thus, the presence of the calibration standard permits absolutequantification of the labeled analytes (and in some cases theirprecursors) in each differentially labeled sample mixed to form thesample mixture.

Because the analyte can be a precursor molecule, in some embodiments,the analytes can be peptides and the identity and absolute amount of oneor more proteins in each of the two or more differentially labeledsamples mixed to form the sample mixture can be determined based uponthe identity and absolute amount of the one or more peptides in each ofthe two or more differentially labeled samples mixed to form the samplemixture.

As previously discussed, this analysis can be repeated one or more timeson selected ions of a different mass to charge ratio to thereby obtainthe absolute amount of one or more other determined analytes in eachsample combined to form the sample mixture. Moreover, as appropriate, acorrection of peak intensity associated with each unique reporter ioncan be performed for naturally occurring, or artificially created,isotopic abundance, as previously discussed.

In some embodiments, methods described herein can be practiced withsupport-bound labeling reagents, wherein each different labeling reagentof the set is support-bound and is cleavable linked to the support bythe reporter moiety through a cleavable linker such that each differentsample can be reacted with a support carrying a different labelingreagent of the set. Exemplary supports have been discussed above in thesection entitled: “Compositions” (Also see FIG. 6). According to themethod, the support can be optionally washed to remove components of thesample that do not react with the reactive group of the labeling reagentafter the analyte has been permitted to react with the support-boundlabeling reagent but before the samples are mixed. Once the analyte hasbeen permitted to react with the labeling reagent to thereby form thelabeled analyte and the washing step is optionally performed, thelabeled analyte(s) can be released from the support by treating thesupport under conditions whereby the cleavable linker is cleaved. Oncecleaved, each of the two or more differentially labeled samples can beoptionally collected, each sample comprising one or more labeledanalytes wherein the labeled analytes associated with a particularsample are identifiable and/or quantifiable by the unique reportermoiety linked thereto. Whether or not they are collected individually,the products of cleavage can be mixed to form a sample mixture.

IV. Mixtures

In some embodiments, this invention pertains to mixtures (i.e. samplemixtures). For example, the mixtures can comprise labeled analytescomprising isotopically encoded isomeric and/or isobaric labels.Exemplary mixtures of labeled analytes and methods for their preparationand/or analysis have been described in the section entitled: “Methodsfor Labeling and Analysis”, set forth above.

The mixture can be formed by mixing all, or a part, of the product oftwo or more labeling reactions wherein each sample is labeled with adifferent labeling reagent of a set of labeling reagents wherein eachlabeling reagent comprises a reporter moiety of unique (gross) mass aswell as a non-encoded detectable label. The unique reporter moiety ofeach different labeling reagent can identify from which labelingreaction each of the two or more labeled analytes is derived (i.e.originated). The non-encoded detectable label can be used to locatelabeled analytes during an electrophoretic separation. Characteristicsof the labeling reagents and labeled analytes associated with thesemethods have been previously discussed.

One or more of the analytes of the mixture can be peptides. One or moreof the analytes of the mixture can be proteins. One or more of theanalytes of the mixture can be peptides and proteins. One or more of theanalytes of the mixture can be nucleic acid molecules. One or more ofthe analytes of the mixture can be carbohydrates. One or more of theanalytes of the mixture can be lipids. One or more of the analytes ofthe mixture can be steroids. One or more of the analytes of the mixturecan be vitamins. One or more of the analytes of the mixture can beprostaglandins. One or more of the analytes of the mixture can be aminoacids. One or more of the analytes of the mixture can be small moleculeshaving a mass of less than 1500 daltons. In some cases the analytes canbe mixtures of various analyte types. For example, the analytes of themixture comprise lipids and steroids; or 2) proteins, peptides, aminoacids, lipids, steroids and carbohydrates). Mixtures can comprise anytype of differentially labeled analytes labeled with the novel labelingreagents disclosed herein including mixtures comprising two or moredifferent analyte types.

For example, the mixtures can comprise at least two differentiallylabeled analytes wherein said mixture is formed by mixing the product ofa labeling reaction of at least two different samples each sample beinglabeled with a different labeling reagent from a set of isotopicallyencoded isomeric and/or isobaric labeling reagents, wherein eachdifferentially labeled analyte of the mixture is represented by formulaI^(A):

including a salt form and/or hydrate form thereof, wherein m and n areeach 0 or 1 provided that m+n=1; The reporter moiety “RP” (as describedin more detail above) comprises a fixed charge or is ionizable in a massspectrometer, wherein said reporter moiety is capable of producing asignature ion in a mass spectrometer upon fragmentation of bond X andwherein the gross mass of each reporter moiety, and its correspondingsignature ion, is different for each different sample depending on whichlabeling reagent of the set was used to label the sample. Thenon-encoded detectable label “DL” (as described in more detail above) isdifferent for each different labeling reagent of the set and eachdifferent non-encoded detectable label is independently detectable ofthe others in the set and wherein each different non-encoded detectablelabel has the same gross mass and same net charge as the othernon-encoded detectable labels of the other labeling reagents of the set.The linker moiety “LK” (as described in more detail above) can be linearor branched, wherein LK links (directly or indirectly) the analyte toboth the reporter moiety and the non-encoded detectable label andwherein the mass of the linker moiety of each different labeling reagentof the set compensates for the difference in gross mass between thereporter moieties for the different labeling reagents of the set suchthat the aggregate gross mass of the combination of the reporter moietyand the linker moiety is the same for each labeling reagent of the set.X (as described in more detail above) is a covalent bond that links thereporter moiety to the linker moiety or to the non-encoded detectablelabel and wherein bond X is fragmentable by application of dissociativeenergy in a mass spectrometer. Y (as described in more detail above) isa covalent bond or linking group that links the analyte to the linkermoiety and wherein Y is optionally fragmentable by application ofdissociative energy in a mass spectrometer, provided that if Y is alinking group its gross mass is the same for every labeling reagent ofthe set. W (as described in more detail above) is a covalent bond orlinking group that links the non-encoded detectable label to the linkermoiety and wherein W is optionally cleavable by application of light,heat and/or chemical reagent(s) and/or fragmentable by application ofdissociative energy in a mass spectrometer, provided that if W is alinking group its gross mass is the same for every labeling reagent ofthe set. Certain fragmentation properties of labeled analyte I^(A) areillustrated in FIG. 3 a.

For example, the mixture can comprise at least one labeled analyterepresented by formula II^(A) or III^(A);

The elements and certain fragmentation properties of labeled analytesII^(A) and III^(A) are illustrated in FIG. 3 b.

In some embodiments, the mixture is prepared by using support-boundlabeling reagents and the labeled analytes are cleaved from the supportto thereby form the mixture. Thus, in some embodiments, this inventionpertains to a mixture comprising at least two differentially labeledanalytes wherein said mixture is formed by mixing the product of alabeling reaction of at least two different samples each sample beinglabeled with a different labeling reagent from a set of isotopicallyencoded isomeric and/or isobaric labeling reagents, wherein eachdifferentially labeled analyte of the mixture is represented by formulaI^(ssAx);

including a salt form and/or hydrate form thereof; wherein m and n areeach 0 or 1 provided that m+n=1. The reporter moiety “RP” (as describedin more detail above) comprises a fixed charge or is ionizable in a massspectrometer, wherein said reporter moiety is capable of producing asignature ion in a mass spectrometer upon fragmentation of bond X andwherein the gross mass of each reporter moiety, and its correspondingsignature ion, is different for each different sample depending on whichlabeling reagent of the set was used to label the sample. Thenon-encoded detectable label “DL” (as described in more detail above) isdifferent for each different labeling reagent of the set, wherein eachdifferent non-encoded detectable label is independently detectable ofthe others in the set and wherein each different non-encoded detectablelabel has the same gross mass and same net charge as the othernon-encoded detectable labels of the other labeling reagents of the set.The linker moiety “LK” (as described in more detail above) can be linearor branched, wherein LK links (directly or indirectly) the analyte toboth the reporter moiety and the non-encoded detectable label andwherein the mass of the linker moiety of each different labeling reagentof the set compensates for the difference in gross mass between thereporter moieties for the different labeling reagents of the set suchthat the aggregate gross mass of the combination of the reporter moietyand the linker moiety is the same for each labeling reagent of the set.X (as described in more detail above) is a covalent bond that links thereporter moiety to the linker moiety or to the non-encoded detectablelabel and wherein bond X is fragmentable by application of dissociativeenergy in a mass spectrometer. Y (as described in more detail above) isa covalent bond or linking group that links the analyte to the linkermoiety and wherein Y is optionally fragmentable by application ofdissociative energy in a mass spectrometer, provided that if Y is alinking group its gross mass is the same for every labeling reagent ofthe set. W (as described in more detail above) is a covalent bond orlinking group that links the non-encoded detectable label to the linkermoiety and wherein W is optionally cleavable by application of light,heat, chemical reagent(s) and/or fragmentable by application ofdissociative energy in a mass spectrometer, provided that if W is alinking group its gross mass is the same for every labeling reagent ofthe set. Q′ is a functional group formed by cleavage of thedifferentially labeled analyte from a solid support. For example Q′ canbe an alkyl amine group, an alkyl hydroxyl group or an alkyl thiolgroup.

For example, the mixture can comprise at least one labeled analyterepresented by formula II^(ssAx) or III^(ssAx);

wherein Q′ is a functional group formed by cleavage of thedifferentially labeled analyte from a solid support. The elements andcertain fragmentation properties of labeled analytes II^(ssAx) andIII^(ssAx) are illustrated in FIG. 3 d.V. Kits

In some embodiments, this invention pertains to kits. The kits cancomprise one or more labeling reagents as described herein as well asone or more other reagents, containers, enzymes, buffers and/orinstructions. For example, the reagents of the kit can be selected toperform an assay for quantifying one or more analytes in two or moredifferent samples. The kits can comprise a set of two or more labelingreagents and one or more other reagents, containers, enzymes, buffersand/or instructions. Two or more of the labeling reagents of a kit canbe isomeric and/or isobaric. The labeling reagents can be isotopicallyencoded.

The labeling reagents of the kit can be any labeling reagent describedherein. For example, the one or more labeling reagents of the kits canbe compounds (including sets of compounds) of the formula: I, II, III,I^(ss), II^(ss), and/or III^(ss), as previously disclosed herein,including isotopically encoded versions thereof.

In some embodiments, the kit can comprise a labeled analyte of formula:I^(A), II^(A), III^(A), I^(ssAx), II^(ssAx), and/or III^(ssAx), aspreviously disclosed herein, including isotopically encoded versionsthereof. Other properties of the labeling reagents of the kits have beendisclosed. In some embodiments, at least one additional reagent of thekit can be a labeled calibration standard comprising a reporter moiety.In some embodiments, the reporter can have a unique mass as comparedwith any of the compound (i.e. labeling reagents) in the kit. The kitscan, for example, be useful for the multiplex analysis of one or moreanalytes in the same sample, or in two or more different samples.

EXAMPLES

Aspects of the present teachings can be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1 Proposed Synthetic Route to Labeling Reagents

With reference to FIGS. 9 a and 9 b, a proposed synthetic route toexemplary labeling reagent III is presented. It is to be understood thatthe same method can be used to prepare exemplary labeling reagent II bysimple substitution of the appropriate fluorescent dye. It also is to beunderstood that the compositions can be prepared with isotopicallyenriched sites by substitution of appropriately encoded startingmaterials.

With reference to FIG. 9 a, compound 10 is reacted with compound 11 inthe presence of sodium hydride (NaH) to thereby produce the productcompound 12. Compound 12 is treated with acetic anhydride (Ac₂O) tothere by form Compound 13. Compound 13 is treated with compound 14(including isotopically encoded versions thereof as appropriate—See forexample, Example 2, below) to thereby produce compound 15.

With reference to FIG. 9 b, compound 15 is then treated with methanol(MeOH) and dicyclohexylcarbodiimide (DCC) to thereby produce compound16. Compound 16 is then treated with hydrochloric acid (HCl) in dioxaneto thereby produce compound 17. Compound 17 is then treated under basicconditions with a non-nucleophilic base, such as triethylamine (Et₃N),and an activated (e.g. activated as an N-hydroxysuccinimide ester (NHSester)) non-encoded detectable label, such as compound 18, to therebyproduce compound 19. Compound 18 is a one possible non-encodeddetectable label and this label is represented in compounds of formulaIII, illustrated herein. An activated form of a non-encoded detectablelabel, such as that represented in compounds of formula II, could alsobe used to form a companion label of a set of isomeric and/or isobariclabeling reagents. Compound 19 is then treated with a strong base, suchas sodium hydroxide (NaOH) to saponify the methyl ester and theresulting carboxylic acid is converted to an N-hydroxysuccinimidyl esterby treatment with N-hydroxysuccinimidyl-trifluoroacetate (NHS-TFA),which is a reagent commonly known to be suitable for convertingcarboxylic acid groups to N-hydroxysuccinimidyl esters (see FIG. 7), tothereby produce labeling reagent 20.

Example 2 Proposed Synthetic Route to Encoded Reporter Molecules (i)Synthesis of BocNH¹³CH₂ ¹³CH₂OH (31)

With reference to FIG. 10 a, BocNH¹³CH₂ ¹³COOH (30, P/N: Aldrich 604992;10 g, 56.44 mmol) was transferred to a 500 mL two-neck round bottomflask equipped with septa, magnetic stir bar and argon input line. Afterflushing the system with argon, dry tetrahydrofuran (THF, P/N: Aldrich,186562; 100 mL) was transferred via cannula using pressure differenceand stirred at room temperature until a clear solution was obtained. Thereaction mixture then cooled to 0° C. and a solution of boran-THFcomplex (BH₃.THF, P/N: Aldrich 176192, 1 M, 197 mL) was added to thereaction mixture, via cannula, over 10 min period. The reaction thencontinued for another 90 minutes (min) at 0° C.

A small aliquot of the reaction mixture was quenched with methanol(MeOH) and analyzed by thin layer chromatography (TLC), which showedcomplete consumption of starting material 30 and formation of theproduct 31 (R_(f)30=0.00, R_(f)31=0.20; 1:1 hexanes-ethyl acetate(EtOAc); TLC developed by heating with 3% (w/v) ninhydrin solution inethanol (EtOH)).

The reaction was quenched (at 0° C.) by slow addition of MeOH (100 mL,added over 20 min). Volatiles were removed under reduced pressure. Theoil so obtained was coevaporated from additional MeOH (50 mL) andpurified by column chromatography (two runs; 120 g Si₂ column Isco.; 85mL/min, 0-10 min 40% EtOAc in hexanes, 10-25 min 90% EtOA hexanes. 18mL-fractions collected; fractions 39-55 had pure product) to giveBocNH¹³CH₂ ¹³CH₂OH (31) as colorless oil (7.40 g, 81%).

(ii) Synthesis of BocNHCH₂CH₂OMs (32)

With reference to FIG. 10 b, to an ice cold solution of 31 (1.15 g, 7.13mmol), Et₃N (2.5 mL, 17.82 mmol) in dichloromethane (DCM, 100 mL) wasadded methanesulfonyl chloride (MsCl, P/N: Fluka 64260, 0.664 mL, 8.55mmol) over 1 min while stirring. After another 15 min at 0° C. thereaction mixture was analyzed by TLC and showed formation of a newproduct (R_(f)31=0.20, R_(f)32=0.42; 1:1 hexanes-EtOAc; TLC developed byheating with 3% (w/v) ninhydrin solution in EtOH).

DCM evaporated and the yellow solid was dissolved in EtOAc (300 mL). TheEtOAc layer was washed with HCl (1 M, 100 mL), followed by brine (50mL×2), dried over Na₂SO₄ and concentrated to give yellow oil, which wasused in the next reaction without purification.

(iii) Synthesis of BocNHCH₂CH₂NHMe (33)

With reference to FIG. 10 c, BocNHCH₂CH₂OMs (32) (1.42 g, 5.94 mmol) wastransferred to a Chem-Glass pressure vessel using minimum amount of THFto which 60 mL of methylamine (MeNH₂, P/N: Aldrich 395056, 2.0 M in THF,120 mmol) solution was added, capped and heated (while stirring) at40-45° C. for 3 h, then at RT for overnight (use safety shield). TLCanalysis (1:1 EtOAc-hexanes) showed complete consumption of 32 andformation of a new product (R_(f)33=0.38; 1:1 DCM-MeOH+1% (v/v) Et₃N;TLC plate was heated first for 5 min to remove Et₃N then developed byheating with 3% (w/v) ninhydrin solution in EtOH). Reaction mixture thenconcentrated to an oil and purified by column chromatography (40 g SiO₂column Isco.; 40 mL/min, Column equilibrated with EtOAc, eluted with 1:1MeOH-DCM+1% Et₃N (v/v). 18 mL-fractions collected; fractions 13-19 hadpure product) to give BocNH¹³CH₂ ¹³CH₂NHMe 33 as colorless oil (0.79 g,76%).). ES-MS (Direct infusion in MeOH) Calculated MH⁺[¹³C₂C₆H₁₈N₂O₂+H]⁺=177.14, observed MH⁺=177.14.

(iv) Synthesis of NH₂CH₂CH₂NH(Me)Fmoc (34)

With reference to FIG. 10 d, to a well stirred solution of BocNH¹³CH₂¹³CH₂NHMe (33, 1.78 g, 10.2 mmol) in acetone (125 mL) is added asolution of Fmoc-OSu (P/N: Advance ChemTech RC8015, 3.46 g, 10.2 mmol in125 mL acetone). The mixture is stirred for 10 min at ambienttemperature. At this point NaHCO₃ (saturated, 25 mL) solution is addedto the reaction mixture, to adjust the pH to 8-9, and stirred vigorouslyfor another 30 min (biphasic reaction). After evaporation of acetone,the product is partitioned between EtOAc (500 mL) and dilute (aq)hydrochloric acid (HCl, 30 mL, 1 M)+30 mL brine. The EtOAc layer is thenfurther washed with dilute HCl (30 mL, 1 M)+30 mL brine, followed by amixture of NaHCO₃ (10 mL)+brine (60 mL) and brine (50 mL×2), dried overNa₂SO₄ and concentrated to give a thick colorless oil.

The oil is then dissolved in DCM (50 mL), HCl solution in dioxane (4 M,50 mL) is added and the reaction mixture is stirred for 30 min atambient temperature. Volatiles are removed under reduced pressure togive a white dry solid, which is washed with 7:3 EtOAc-hexanes to giveNH₂ ¹³CH₂ ¹³CH₂N(Fmoc)Me 34 as a solid.

(v) Synthesis of the a Partial Encoded Reporter/Linker Moiety (35)

With reference to FIG. 11, to a solution of 34 (3 eqv) andN,N′-diisopropylethylamine (DIEA, 6 eqv) in DCM, trityl-Cl resin(Advance ChemTech, P/N SC5028, 1 eqv) is added as a solid and mixed for30 min. The resin is then filtered and washed with DMF. The resin isthen treated with a 20% solution of piperidine in N,N′-dimethylformamide(DMF) for 5 min, and washed with DMF.

To a solution of N-methyl piperazine acetic acid (Chess GmbH, P/N 2022,3 eqv) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate HATU (3 eqv) in N-Methyl-2-pyrrolidinone (NMP), isadded DIEA (9 eqv). This is mixed for 1 min and the added to the resin.After 30 min coupling, the resin is washed with NMP followed byacetonitrile.

Product (35) is cleaved from the resin using 15% TFA in DCM to give thecompound as a TFA salt.

It is to be understood encoded N-methyl piperazine acetic acid (See forExample US Published Patent Applications US 2005-0148774 A1 and US2005-01448773 A1 for exemplary syntheses of encoded N-methyl piperazineacetic acid) and non-encoded NH₂CH₂CH₂N(Fmoc)Me 34′ (FIG. 12) can besubstituted in the reaction synthesis to thereby produce a correspondingversion of compound 35 (identified as 35′ in FIG. 12), wherein thereporter comprises the isotopically enriched sites (See for example, thealternative synthesis illustrated in FIG. 12). By judicious selection ofstarting materials, essentially any desired encoded version of compound35 can be prepared using the method(s) illustrated.

In some embodiments, the encoded N-methyl piperazine acetic acidmoieties can be selected from compounds 36 or 37 represented byformulas:

wherein the * indicates substitution of a ¹³C for a ¹²C.

Example 3 Alternative Proposed Synthetic Route to Labeling Reagents

With reference to FIGS. 13 and 14, an alternative synthetic route to thelabeling reagents is illustrated wherein the linker that cleavably linksthe non-encoded detectable label to the remainder of the labelingreagent is inverted as compared with the description in Example 1,above. With reference to FIG. 13 a, the fluorescent dye 40 (it is to beunderstood that one could choose the fluorescent dye that leads tocompounds of formula II as disclosed herein) is reacted with compound 41in the presence of dicyclohexylcarbodiimide (DCC) to thereby producecompound 42. The tert-butyloxycarbonyl (t-boc or Boc) group of compound42 is then removed by treatment with trifluoroacetic acid (TFA) tothereby produce compound 43 as its TFA salt. With reference to FIG. 13a, compound 43 is also illustrated in a shorthand form as compound 43′which from is used in the illustration in FIG. 14.

With reference to FIG. 13 b, compound 44 (which can be preparedaccording to: Fadeev, Evgeny A.; Luo, Minkui; Groves, John T. Synthesisand structural modeling of the amphiphilic siderophore rhizobactin-1021and its analogs. Bioorganic & Medicinal Chemistry Letters (2005),15(16), 3771-3774) is reacted with compound 35 (it is to be understoodthat depending on the desired reagent, various encoded versions or anon-encoded version of compound 35 can be used) in the presence of anon-nucleophilic organic base, such as triethylamine (Et₃N) orN,N′-diisopropylethylamine (DIEA), to thereby produce compound 45.Compound 45 is reacted with benzyl bromide (BnBr) in the presence of theinorganic base; cesium carbonate (CsCO₃) to thereby form compound 46.The t-boc group of compound 46 is then removed by treatment with TFA tothereby produce compound 47. Compound 47 is then reacted treated withHATU in the presence of a non-nucleophilic organic base, such as DIEA,and then reacted with compound 43′ to thereby produce compound 48.Compound 48 is then treated with hydrogen (H₂) and Palladium (Pd)catalyst to thereby convert the benzyl ester to a carboxylic acid groupwhich is reacted with N-hydroxysuccinimidyl-triflouroacetate (NHS-TFA,compound 50) to thereby form the NHS-ester of the labeling reagent 49.

Example 4 Proposed Synthetic Route to Non-Encoded Detectable Labels

The fluorescent dyes used as non-encoded detectable labels can beavailable dyes and/or dyes that are modified from commercially availabledyes. For example, with reference to FIG. 14, the compositions disclosedherein with the general formula III, comprise TAMRA moiety, whereinTAMRA is a dye commercially available from various sources.

In order to prepare a fluorescent dye comprising the same mass andcharge as TAMRA, FIG. 14 also illustrates a proposed synthetic route tothe dye used with respect to compounds with general formula II. Theproposed synthesis follows a well known synthetic pathway wherein anN-ethyl version of phenolic compound is substituted with its N-methylderivative 60 to thereby produce the desired dye 65 as its bis-TFA salt.

With reference to FIG. 14, dye 65 can be prepared by treating a mixtureof compound 60 and 61 with polyphosphoric acid under high temperaturefor an extended period of time. Those of skill in the art of fluorescentdye manufacture will appreciate how to perform this meld. The reactionproduces compounds 62 and 63, wherein compound 63 is isolated andconverted to salt form 64. Compound 64 can then be treated withtrifluoroacetic acid/triethylamine to thereby produce thebis-trifluoroactate 65.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications and equivalents, as will beappreciated by those of skill in the art.

1. The compound:

and any salt form thereof, wherein: RG is:

and wherein X′ is O or S and each X″ is independently of the other F,Cl, Br or I.
 2. The compound:

and any salt form thereof, wherein: RG is:

and wherein X′ is O or S and each X″ is independently of the other F,Cl, Br or I.